Liposomes Useful for Drug Delivery

ABSTRACT

The present invention provides liposome compositions containing substituted ammonium and/or polyanion, and optionally with a desired therapeutic or imaging entity. The present invention also provide methods of making the liposome compositions provided by the present invention.

STATEMENT OF PRIORITY

This application is a continuation of U.S. application Ser. No.15/896,389, filed Feb. 14, 2018, which is a continuation of U.S.application Ser. No. 15/227,561, filed Aug. 3, 2016, which is acontinuation of U.S. application Ser. No. 14/879,358, filed Oct. 9,2015, which is a continuation of U.S. application Ser. No. 14/632,422,filed Feb. 26, 2015, which is a continuation of U.S. application Ser.No. 14/175,365, filed Feb. 7, 2014, which is a continuation of U.S.application Ser. No. 13/654,373, filed Oct. 17, 2012, which is acontinuation of U.S. application Ser. No. 13/416,204, filed Mar. 9,2012, which is a continuation of U.S. application Ser. No. 11/121,294,filed May 2, 2005, which claims benefit of priority of the U.S.Provisional Patent Application No. 60/567,921 filed on May 3, 2004, allof which are incorporated herein by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

This invention relates generally to the field of liposomes, and morespecifically to liposome compositions useful for delivery of therapeuticor diagnostic entities.

BACKGROUND OF THE INVENTION

Liposomes, or lipid bilayer vesicles, have been used or proposed for usein a variety of applications in research, industry, and medicine,particularly for the use as carriers of diagnostic or therapeuticcompounds in vivo. See, for example: Lasic, D. Liposomes: from physicsto applications. Elsevier, Amsterdam, 1993. Lasic, D, andPapahadjopoulos, D., eds. Medical Applications of Liposomes. Elsevier,Amsterdam, 1998. Liposomes are usually characterized by having aninterior space sequestered from an outer medium by a membrane of one ormore bilayers forming a microscopic sack, or vesicle. Bilayer membranesof liposomes are typically formed by lipids, i.e. amphiphilic moleculesof synthetic or natural origin that comprise spatially separatedhydrophilic and hydrophobic domains. See Lasic D., 1993, supra. Bilayermembranes of the liposomes can be also formed by amphiphilic polymersand surfactants (polymerosomes, niosomes). A liposome typically servesas a carrier of an entity such as, without limitation, a chemicalcompound, a combination of compounds, a supramolecular complex of asynthetic or natural origin, a genetic material, a living organism, aportion thereof, or a derivative thereof, that is capable of having auseful property or exerting a useful activity. For this purpose, theliposomes are prepared to contain the desired entity in aliposome-incorporated form. The process of incorporation of a desiredentity into a liposome is often referred to as “loading”. Theliposome-incorporated entity may be completely or partially located inthe interior space of the liposome, within the bilayer membrane of theliposome, or associated with the exterior surface of the liposomemembrane. The incorporation of entities into liposomes is also referredto as encapsulation or entrapment, and these three terms are used hereininterchangingly with the same meaning. The intent of the liposomalencapsulation of an entity is often to protect the entity from thedestructive environment while providing the opportunity for theencapsulated entity to exert its activity mostly at the site or in theenvironment where such activity is advantageous but less so in othersites where such activity may be useless or undesirable. This phenomenonis referred to as delivery. For example, a drug substance within theliposome can be protected from the destruction by enzymes in the body,but become released from the liposome and provide treatment at the siteof disease.

Ideally, such liposomes can be prepared to include the desired compound(i) with high loading efficiency, that is, high percent of encapsulatedentity relative to the amount taken into the encapsulation process; (ii)high amount of encapsulated entity per unit of liposome bilayermaterial; (iii) at a high concentration of encapsulated entity, and (iv)in a stable form, i.e., with little release (leakage) of an encapsulatedentity upon storage or generally before the liposome appears at the siteor in the environment where the liposome-entrapped entity is expected toexert its intended activity.

Therefore, there is a need in the art to provide various liposomecompositions that are useful for delivery of a variety of compounds,especially therapeutic, diagnostic, or imaging entities.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that substitutedammonium and polyanion are useful for loading and retaining entitiesinside liposomes. Accordingly the present invention provides methods andliposome compositions useful for delivery of a variety of entities,especially therapeutic entities, that is, entities useful in thediagnosis, prognosis, testing, screening, treatment, or prevention of anundesirable condition, e.g., a disease, in living organism, such as ahuman, a plant, or an animal.

In one embodiment, the present invention provides a compositioncomprising a liposome in a medium, wherein the inside of the liposomecontains a substituted ammonium

wherein each of R₁, R₂, R₃, and R₄ is independently a hydrogen or anorganic group having, inclusively, in totality up to 18 carbon atoms,wherein at least one of R₁, R₂, R₃, and R₄ is an organic group, whereinthe organic group is independently a hydrocarbon group having up to 8carbon atoms, and is an alkyl, alkylidene, heterocyclic alkyl,cycloalkyl, aryl, alkenyl, or cycloalkenyl group or ahydroxy-substituted derivative thereof, optionally including within itshydrocarbon chain a S, O, or N atoms, forming an ether, ester,thioether, amine, or amide bond, wherein at least three of R₁, R₂, R₃,and R₄ are organic groups, or the substituted ammonium is a stericallyhindered ammonium, such as, for example, where at least one of theorganic groups has a secondary or tertiary carbon atom directly linkedto the ammonium nitrogen atom. Preferably, the substituted ammoniumcompound encapsulated into liposomes has a negative logarithm of theacidic (deprotonation) dissociation constant (pKa) of at least about8.0, at least about 8.5, at least about 9.0, at least 9.5, or at least10.0, as determined in an aqueous solution at ambient temperature.

In another embodiment, the present invention provides a compositioncomprising a liposome in a medium, wherein the inner space of theliposome contains a polyanion and wherein the polyanion is apolyanionized polyol or a polyanionized sugar. The liposome preferablycontains a transmembrane gradient capable of effecting the loading of anentity into the liposome. In one embodiment, the transmembrane gradientis a gradient of an ammonium, a quarternary ammomium, or a primary,secondary, or tertiary substituted ammonium compound having in a dilutedaqueous solution at ambient temperature a negative logarithm of theacidic (deprotonation) dissociation constant (pKa) of at least about8.0, at least about 8.5, at least about 9.0, at least 9.5, or at least10.0. The liposome optionally contains an entrapped entity, for example,a therapeutic, a detectable marker, or a globally cationic organicmolecule.

In yet another embodiment, the composition provided by the presentinvention further comprises an entity encapsulated in the liposomes ofthe present invention. Preferably, the entity is encapsulated within theinner space of the liposome. For example, the inner space of theliposome further comprises an anti-neoplastic therapeutic and whereinthe toxicity level of the composition to a subject is at least equal toor less than the toxicity level of the anti-neoplastic therapeuticadministered to the subject without the composition.

In yet another embodiment, the composition provided by the presentinvention is a liposome composition comprising a camptothecin compound.The composition has an anticancer activity at least two times, fourtimes, or ten times higher than the camptothecin compound similarlyadministered in the absence of the composition, while the toxicity ofthe composition does not exceed, is at least two times, or at least fourtimes lower than the toxicity of the camptothecin compound similarlyadministered in the absence of the composition. In a one embodiment, thecamptothecin compound is a pro-drug, and is contained in the liposome ofat least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.5 mg, orat least 1 mg_per 1 mg of the liposome membrane materials, e.g., lipids.The camptothecin compound is preferably encapsulated in the liposomesubstantially within the inner space of the liposome. In one instance,the camptothecin compound is irinotecan (CPT-11).

In yet another embodiment, the composition provided by the presentinvention is a liposome composition of a vinca alkaloid or a derivativethereof. The composition has the 24-hour drug retention within theliposome after 24 hours exposure in the blood of a mammal in vivo of atleast 50%, at least 60%, or at least 70% of the original drug load. Thevinca alkaloid or a derivative thereof is preferably encapsulated in theliposome substantially within the inner space of the liposome. Oneexample of the mammal is a rat. Exemplary vinca alkaloids andderivatives are vincristine, vinblastine, and vinorelbine.

In still another embodiment, the present invention provides a method ofencapsulating an entity into a liposome. The method comprises contactingthe liposomes of the present invention with an entity, e.g., therapeuticor detectable entity. Preferably, the contacting is performed under theconditions when the concentration of substituted ammonium or a polyanionof the present invention in the medium is lower than that in the innerspace of the liposomes. In one embodiment, the liposome composition iscontacted with an entity in an aqueous medium.

In still another embodiment, the present invention provides a method ofencapsulating an entity into a liposome. The method comprises contactingthe liposome-containing composition of the present invention with apre-entity, wherein the pre-entity is capable of being converted to anentity under a condition, and providing the condition inside theliposome whereby converting the pre-entity to the entity inside theliposome. In one case, the entity is an organic compound, and thepre-entity is a basic derivative thereof.

In still another embodiment, the present invention provides a kit formaking liposome-encapsulated entities. The kit comprises a containerwith a liposome of the present invention, and, optionally, a containercontaining an entity, and/or instructions for a user, e.g. toencapsulate an entity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows blood pharmacokinetics of the liposome lipid (circles) andthe drug (triangles) after i.v. bolus administration of CPT-11-loadedliposomes to a rat. The liposomes are loaded using TEA-Pn method (SeeExample 9).

FIG. 2 shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration of the liposomeloaded with CPT-11 using TEA-Pn method (See Example 9).

FIG. 3 shows antitumor efficacy of freeCPT-11 and liposomal CPT-11against BT-474 human breast cancer xenografts in nude mice. “Control”designates the mice treated with drug- and liposome-free vehicle only.(See Example 10).

FIG. 4 shows the dynamics of the animals' body weights during thetreatment of BT-474 tumor-bearing nude mice with free CPT-11 orliposomal CPT-11. “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 10).

FIG. 5 shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration of the liposomeloaded with CPT-11 using TEA-SOS method. (See Example 14).

FIG. 6 shows antitumor efficacy of free and liposomal CPT-11 againstHT-29 human colon cancer xenografts in nude mice. The on-panel captionindicates the drug loading method and the administered dose perinjection. “Saline control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 15).

FIG. 7 shows the dynamics of the animals' body weights during thetreatment of HT-29 tumor-bearing nude mice with free or liposomalformulations of CPT-11. The error bars represent standard deviation ofthe data. “Saline control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 15).

FIG. 8A shows blood pharmacokinetics of the liposome lipid after i.v.bolus administration of Topotecan-loaded liposomes to a rat. Theon-panel caption indicates the drug loading method and the drug contentof the liposomes. (See Example 24).

FIG. 8B shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration of the liposomesloaded with Topotecan The on-panel caption indicates the drug loadingmethod and the drug content of the liposomes. (See Example 24).

FIG. 9 shows the in vitro cytotoxicity of free, liposomal, orHER2-targeted immunoliposomal Topotecan (TEA-Pn method) against SKBr-3breast carcinoma cells. (See Example 27).

FIG. 10 shows the in vitro cytotoxicity of free, liposomal, orHER2-targeted immunoliposomal Topotecan (TEA-SOS method) against SKBr-3breast carcinoma cells. (See Example 32).

FIG. 11 shows antitumor efficacy of various Topotecan (TPT) formulationsagainst BT-474 human breast cancer xenografts in nude mice. “Salinecontrol” designates the mice treated with drug- and liposome-freevehicle only. (See Example 29).

FIG. 12 shows the dynamics of the animals' body weights during thetreatment of BT-474 tumor-bearing nude mice with free Topotecan (TPT),liposomal Topotecan (Ls-TPT), or anti-HER2 immunoliposomal Topotecan (F5ILs-TPT). “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 29).

FIG. 13A shows antitumor efficacy of Topotecan formulations againstBT-474 human breast cancer xenografts in nude mice. Free Topotecan (FreeTPT) or liposomal Topotecan (Ls-TPT) were administered at one-eighth oftheir maximum tolerated doses. Error bars represent standard deviationof the data. “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 31).

FIG. 13B shows antitumor efficacy of Topotecan formulations againstBT-474 human breast cancer xenografts in nude mice. Free Topotecan (FreeTPT) or liposomal Topotecan (Ls-TPT) were administered at one-fourth oftheir maximum tolerated doses. Error bars represent standard deviationof the data. “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 31).

FIG. 13C shows antitumor efficacy of Topotecan formulations againstBT-474 human breast cancer xenografts in nude mice. Free Topotecan (FreeTPT) or liposomal Topotecan (Ls-TPT) were administered at one-half oftheir maximum tolerated doses. Error bars represent standard deviationof the data. “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 31).

FIG. 13D shows antitumor efficacy of Topotecan formulations againstBT-474 human breast cancer xenografts in nude mice. Free Topotecan (FreeTPT) or liposomal Topotecan (Ls-TPT) were administered at their maximumtolerated doses. Error bars represent standard deviation of the data.“Control” designates the mice treated with drug- and liposome-freevehicle only. (See Example 31).

FIG. 14 shows the dynamics of the average body weights during thetreatment of BT-474 tumor-bearing nude mice with free Topotecan (FreeTPT) or liposomal Topotecan (Ls-TPT) administered at their maximumtolerated doses. “Control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 31).

FIG. 15 shows the cytotoxicity of free 6-(3-aminopropyl)-ellipticine(Free AE), liposomal 6-(3-aminopropyl)-ellipticine (Ls-AE), orHER2-targeted immunoliposomal 6-(3-aminopropyl)-ellipticine (F5 ILs-AE))against BT-474 breast carcinoma cells in vitro. (See Example 35).

FIG. 16 shows the in vitro cytotoxicity of free6-(3-aminopropyl)-ellipticine (Free APE), liposomal6-(3-aminopropyl)-ellipticine (Ls-APE), or EGFR-targeted immunoliposomal6-(3-aminopropyl)-ellipticine (C225-ILs-APE) against breast carcinomacells with low (MCF-7) or high (MDA-MB468) expression of EGF receptor.(See Example 36).

FIG. 17 shows blood pharmacokinetic attributes of the liposomallyformulated 6-(3-aminopropyl)ellipticine (APE): blood pharmacokinetics ofthe liposome lipid (Panel A, open circles), the drug (Panel A, filledcircles), and the dynamics of drug-to-liposome lipid ratio (Panel B)after i.v. bolus administration of APE liposomes to a rat. (See Example37).

FIG. 18 shows blood pharmacokinetic attributes of vinorelbine formulatedinto liposomes (Ls-VRB), and anti-HER2 immunoliposomes (F5-ILs-VRB):blood pharmacokinetics of the liposome lipid (Panel A), the drug (PanelB), and the dynamics of drug-to-liposome lipid ratio (Panel C) afteri.v. bolus administration of vinorelbine liposomes to a rat. (SeeExample 43).

FIG. 19 shows blood pharmacokinetics of the liposome lipid after i.v.bolus administration of vinorelbine-loaded liposomes to a rat. Theliposomes are loaded using pre-entrapped triethylammonium dextransulfate(DS-TEA), ammonium dextransulfate (DS-A), or ammonium sulfate (S-A).(See Example 44).

FIG. 20 shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration of the liposomesloaded with vinorelbine using pre-entrapped triethylammoniumdextransulfate (DS-TEA), ammonium dextransulfate (DS-A), or ammoniumsulfate (S-A). (See Example 44).

FIG. 21 shows blood pharmacokinetics of the liposome lipid after i.v.bolus administration of vinorelbine-loaded liposomes to a rat. Theliposomes are loaded using pre-entrapped triethylammoniumsucroseoctasulfate (TEA-SOS) and have the mean size as indicated at theon-panel caption. (See Example 45).

FIG. 22 shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration ofvinorelbine-loaded liposomes. The liposomes are loaded usingpre-entrapped triethylammonium sucrooctasulfate (TEA-SOS) and have themean size as indicated at the on-panel caption. (See Example 45).

FIG. 23 shows blood pharmacokinetics of the liposome lipid in a ratafter i.v. bolus administration of vinorelbine formulated into liposomes(Ls-VRB) or anti-HER2 immunoliposomes (F5-ILs-VRB) using TEA-SOS method.(See Example 46).

FIG. 24 shows the dynamics of drug-to-liposome lipid ratio in the bloodof a rat in vivo following i.v. bolus administration of vinorelbineformulated into liposomes (Ls-VRB) or anti-HER2 immunoliposomes(F5-ILs-VRB) using TEA-SOS method. (See Example 46).

FIG. 25 shows the in vitro cytotoxicity of free vinorelbine (free VRB),liposomal vinorelbine (Ls-VRB), or HER2-targeted immunoliposomalvinorelbine (F5-Ils-VRB) against HER2-overexpressing human breast cancercells MDA-MB-453. (See Example 48).

FIG. 26 shows the in vitro cytotoxicity of free vinorelbine (free VRB),liposomal vinorelbine (Ls-VRB), or HER2-targeted immunoliposomalvinorelbine (F5-Ils-VRB) against HER2-overexpressing CaLu-3 humannon-small cell lung cancer cells. (See Example 49).

FIG. 27 shows the in vitro cytotoxicity of free vinorelbine (free VRB),liposomal vinorelbine (Ls VRB/SOS-TEA), or HER2-targeted immunoliposomalvinorelbine (F5-ILs VRB/SOS-TEA) against HER2-overexpressing humanbreast cancer cells SKBr-3. (See Example 50).

FIG. 28 shows antitumor efficacy of the free vinorelbine (free VRB) orliposomal vinorelbine (Ls VRB) against HT-29 human colon cancerxenografts in nude mice. “Saline” designates the mice treated with drug-and liposome-free vehicle only. Error bars represent standard deviationof the data. (See Example 51).

FIG. 29 shows the dynamics of the average body weights during thetreatment of HT-29 tumor-bearing nude mice with free vinorelbine (freeVRB), liposomal vinorelbine (Ls VRB), or vehicle only (saline). Errorbars represent standard deviation of the data. (See Example 51).

FIG. 30 shows antitumor efficacy of the free vinorelbine (free VRB) orliposomal vinorelbine (Ls VRB) in a syngeneic C-26 murine coloncarcinoma model. The dose of the drug per injection was as indicated onthe on-panel caption. Error bars represent standard deviation of thedata. “Saline” designates the mice treated with drug- and liposome-freevehicle only. (See Example 52).

FIG. 31 shows the dynamics of the average body weights during thetreatment of mice bearing syngeneic C-26 murine colon carcinoma tumorswith various doses of free vinorelbine (free VRB), liposomal vinorelbine(Ls VRB), or with vehicle only (saline). The dose of the drug perinjection was as indicated on the on-panel caption. (See Example 52).

FIG. 32 shows antitumor efficacy of the free vinorelbine (Free drug) orscFv F5-conjugated, anti-HER2 immunoliposomal vinorelbine prepared by aTEA-SOS method (F5-ILs-VRB TEA-SOS), anti-HER2 immunoliposomalvinorelbine prepared y a TEA-Pn method (F5-ILs-VRB TEA-Pn) againstHER2-overexpressing human breast carcinoma (BT-474) xenografts in nudemice. “Saline control” designates the mice treated with drug- andliposome-free vehicle only. (See Example 53).

FIG. 33 shows the dynamics of the average body weights during thetreatment of mice bearing HER2-overexpressing human breast carcinoma(BT-474) xenografts with free vinorelbine, scFv F5-conjugated, anti-HER2immunoliposomal vinorelbine prepared using a TEA-SOS method, anti-HER2immunoliposomal vinorelbine prepared by a TEA-Pn method, or with vehicleonly. For explanation of the symbols, see the caption to FIG. 32. (Seealso Example 53).

FIG. 34 shows antitumor efficacy of the free vinorelbine (Free drug) orscFv F5-conjugated, anti-HER2 immunoliposomal vinorelbine prepared usingvarious amounts of PEG-lipid against HER2-overexpressing human breastcarcinoma (BT-474) xenografts in nude mice. The error bars are standarddeviation of the data. “Vehicle control” designates the mice treatedwith drug- and liposome-free vehicle only. (See Example 54).

FIG. 35 shows antitumor efficacy of the free vinorelbine (free NAV),liposomal vinorelbine (NAV Lip), or FC225Fab′-conjugated,anti-EGFR-immunoliposomal vinorelbine (C225-NAV Lip) againstEGFR-overexpressing human glioblastoma (U87) xenografts in nude mice.“Saline” designates the mice treated with drug- and liposome-freevehicle only. (See Example 55).

FIG. 36 shows blood pharmacokinetics of the liposome lipid and thedynamics of the drug/liposome lipid ratio in the blood of a rat afteri.v. bolus administration of doxorubicin formulated into liposomes usingtriethylammonium sulfate method. (See Example 56).

FIG. 37 shows antitumor efficacy of the liposomal doxorubicin (Ls-Dox),or scFv F5-conjugated, anti-HER2 immunoliposomal doxorubicin (F5ILs-Dox) prepared using various amounts of PEG-lipid againstHER2-overexpressing human breast carcinoma (BT-474) xenografts in nudemice. The on-panel caption shows the amount of PEG-lipid expressed inmol. % of liposome phospholipids. “Saline control” designates the micetreated with drug- and liposome-free vehicle only. (See Example 57).

FIG. 38 shows blood pharmacokinetics of liposomal vinblastine in a rat.(See Example 58).

FIG. 39 shows the dynamics of the drug/liposome lipid ratio in the bloodof a rat after i.v. bolus administration of liposomal vinblastine. (SeeExample 58).

FIG. 40 shows the in vitro cytotoxicity of free vincristine (Free VCR),liposomal vincristine (Ls-VCR), or HER2-targeted immunoliposomalvincristine (F5-ILs-VCR) against HER2-overexpressing human breast cancercells SKBr-3. (See Example 61).

FIG. 41 shows blood pharmacokinetics of the liposome lipid in a ratafter i.v. bolus administration of vincristine formulated into liposomesof different average size (indicated on the on-panel caption). (SeeExample 62).

FIG. 42 shows the dynamics of the drug/liposome lipid ratio in the bloodof a rat after i.v. bolus administration of vincristine formulated intoliposomes of different average size (indicated on the on-panel caption).(See Example 62).

FIG. 43 shows antitumor efficacy of the free vincristine (free VCR),liposomal vincristine prepared by triethylammonium citrate method(Ls-VCR Citrate), liposomal vincristine prepared by triethylammoniumsucrooctasulfate method (Ls-VCR SOS), or scFv F5-conjugated, anti-HER2immunoliposomal vincristine prepared by triethylammoniumsucrooctasulfate method (F5 ILs-VCR SOS) against HER2-overexpressinghuman breast carcinoma (BT-474) xenografts in nude mice. “Salinecontrol” designates the mice treated with drug- and liposome-freevehicle only. (See Example 64).

FIG. 44 shows the dynamics of the average body weights during thetreatment of mice bearing HER2-overexpressing human breast carcinoma(BT-474) xenografts with free vincristine (free VCR), liposomalvincristine prepared by triethylammonium citrate method (Ls-VCRCitrate), liposomal vincristine prepared by triethylammoniumsucrooctasulfate method (Ls-VCR SOS), scFv F5-conjugated, anti-HER2immunoliposomal vincristine prepared by triethylammoniumsucrooctasulfate method (F5 ILs-VCR SOS), or with vehicle only (salinecontrol). (See Example 64).

FIG. 45 shows antitumor efficacy of the free vincristine (vincristine),liposomal vincristine (nt-vcr), or C225 Fab′-conjugated, anti-EGFRimmunoliposomal vincristine (C225-vcr) against EGFRvIII-overexpressinghuman brain cancer (U87) xenografts in nude mice. “Saline” designatesthe mice treated with drug- and liposome-free vehicle only. (See Example65).

FIG. 46 shows blood pharmacokinetics of CPT-11 and the dynamics of thepercentage of CPT-11 present in the active (lactone) form in the bloodof a rat after i.v. bolus administration of liposomal CPT-11. (SeeExample 69).

FIG. 47 shows blood pharmacokinetics of CPT-11 and the dynamics of thepercentage of CPT-11 present in the active (lactone) form in the bloodof a rat after i.v. bolus administration of CPT-11 solution (freeCPT-11). (See Example 69).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates in general to methods and liposomecompositions useful for delivery a variety of entities, especiallytherapeutics and imaging agents. It is the discovery of the presentinvention that substituted ammonium and polyanion are useful for loadingand retaining the entities, e.g., compound, inside liposomes.Accordingly, the present invention provides liposome compositions andkits containing substituted ammonium and/or polyanion and methods ofmaking these liposome compositions.

According to one feature of the present invention, it provides acomposition of liposomes containing within its inner space one or moresubstituted ammonium compounds of a formula

wherein each of R₁, R₂, R₃, and R₄ is independently a hydrogen or anorganic group, and wherein at least one of R₁, R₂, R₃, and R₄ is anorganic group, such as, an alkyl, alkylidene, heterocyclic alkyl,cycloalkyl, aryl, alkenyl, or cycloalkenyl group, a hydroxy-substitutedderivative thereof, optionally including within its hydrocarbon chain aS, O, or N atoms, e.g., forming an ether (including an acetal or ketal),ester, sulfide (thioether), amine, or amide bond therein. If less thanthree of R₁, R₂, R₃, and R₄ are organic groups, then, according to theinvention, at least one, and preferably two, of the organic groups has asecondary or tertiary carbon atoms (i.e., carbon atoms having 2 or 3carbon-carbon bonds, respectively) directly linked to the ammoniumnitrogen, i.e., the substituted ammonium is a sterically hinderedammonium. Generally, the presence of titratable ammonium, such asunsubstituted ammonium ion (NH₄ ⁺), as well as primary and secondarystraight chain alkylammonium ions in the inner space of the liposome ofthe present invention is known to provide for enhanced encapsulation ofweak amphiphilic bases, for example, via a mechanism of “active”,“remote”, or “transmembrane gradient-driven” loading (Haran, et al.,Biochim. Biophys. Acta, 1993, v. 1152, p. 253-258; Maurer-Spurej, etal., Biochim. Biophys. Acta, 1999, v. 1416, p. 1-10). However theseammonia compounds possess hydrogen atoms that easily enter intoreactions of nucleophilic substitution, and otherwise react chemicallywith the liposome-entrapped entities, and therefore are capable ofimpairing the chemical integrity of the entities during or after theliposome loading (entrapment) process. Thus, it is desirable for anentrapped substituted ammonium compound to be more chemically inert,lacking chemical functions which are unstable or readily reactive withthe liposome components, that may include an encapsulated entity.Unexpectedly, we discovered that liposome compositions comprising withintheir inner space a substituted tertiary and quaternary ammonium that donot have a substitutable hydrogen, or a sterically hindered primary orsecondary ammonium, in which the access to an ammonium hydrogen atom issterically hindered by a neighbor bulky organic group, such as havingone or two secondary or tertiary carbon atoms linked to the ammoniumnitrogen, show not only outstanding entity-loading capacity, but alsoimproved stability of the liposome-entrapped entity, e.g., a drug,against premature release from the liposome in the living body.

In one embodiment, the liposome-entrapped substituted ammonium compoundis pharmaceutically inert, that is, does not elicit an adversephysiological response when administered to a living subject, e.g. ahuman or an animal, within an amount of the liposome membrane materialthat is sufficient to deliver an effective dose of theliposome-entrapped entity. In another embodiment, the substitutedammonium of the present invention has an acceptable level of toxicity toa subject. Usually an acceptable level of toxicity means that the toxicdose, e.g., a maximum tolerated dose (MTD), or a dose causing 50%lethality (LD50) of the substituted ammonium of the present invention isat least twice, at least four times, at least eight times, or at leastten times higher than the toxic dose of a liposome-entrapped entity,e.g., drug, loaded inside the liposomes of the present invention. Forexample, triethylammonium sulfate has an acceptable level of toxicityaccording to the present invention since its LD50 is about 40 timeshigher than the LD50 of doxorubicin, an anti-cancer drug. The toxicitylevels or physiological responses of substituted ammoniums, as well asof the entities of interest, if not already known, can be readilyestablished via routine techniques well known by persons skilled in thebiomedical art. See, for example, S. C. Gad. Drug Safety Evaluation,Wiley, New York, 2002. One method of quantifying the toxicity of freeand/or liposomally formulated drug is described in Example 16 herein.

In one preferred embodiment, the substituting organic groups among R₁,R₂, R₃, or R₄ are of the size and physico-chemical properties sufficientto ensure that the substituted ammonium forms in aqueous environmentsubstantially a true (molecular) solution, but not micelles, bilayers,or similar self-assembled structures. Therefore, the substitutedammonium of the present invention preferably has little or substantiallyno distribution into the bilayer portion of liposomes, thereforeminimizing the risk of destabilization, solubilization, orpermeabilization of the liposomes entrapping the substituted ammonium.

The organic group of the substituted ammonium is typically a hydrocarboncontaining, inclusively, up to 8 carbon atoms, up to 6 carbon atoms, orup to 4 carbon atoms, and in totality, the substituting groups contain,inclusively, up to 18, up to 16, up to 12, or up to 9 carbon atoms.These substituting hydrocarbon groups include any combination ofinterlinked primary, secondary, or tertiary carbon atoms, as well ascycloalkyl groups being linked at their termini directly to the ammoniumnitrogen to form a heterocycle, or to a carbon atom of an ammoniumhydrogen-substituting group. These substituted alkyl groups can alsoinclude heteroatoms, e.g., oxygen, nitrogen, or sulfur in their carbonchains forming a functional group, e.g., ether, acetal, amine, orsulfide group, as well as forming a functional group, e.g., hydroxylgroup, linked to the alkyl carbon chain. Examples of the organic groupof the present invention include, without any limitation, alkyls,alkylidenes, heterocyclic alkyls, cycloalkyls, aryls, alkenyls,cycloalkenyls, or hydroxy-substituted derivatives thereof, e.g., ahydroxy-substituted alkylidene forming a ring inclusive of N in thesubstituted ammonium.

In another embodiment, the substituted ammonium is: a heterocyclicammonium, i.e. an ammonium wherein at least two of R₁, R₂, R₃, or R₄form a ring; a sterically hindered primary ammonium; or a stericallyhindered secondary ammonium. In general, a sterically hindered primaryor secondary ammonium includes any substituted ammonium with one or twoof the R₁, R₂, R₃, and R₄ substituted with alkyl groups that stericallycrowd the molecule, e.g., any substituted ammonium with one or two ofthe R₁, R₂, R₃, and R₄ substituted with one or two cycloalkyl groups oralkyl groups having at least one secondary or tertiary alkyl carbon atomlinked to the nitrogen of the substituted ammonium. Examples of suchheterocyclic, sterically hindered primary ammoniums, and stericallyhindered secondary ammonium include, without any limitation,isopropylethylammonium, isopropylmethylammonium, diisopropylammonium,tert-butylethylammonium, dicychohexylammonium, protonized forms ofmorpholine, pyridine, piperidine, pyrrolidine, piperazine,tert-bulylamine,2-amino-2-methylpropanol-1,2-amino-2-methyl-propandiol-1,3, andtris-(hydroxyethyl)-aminomethane. These substituted ammonium compoundsare generally commercially available in the form of various salts, orare readily prepared from their corresponding amines by neutralizationwith acids.

In yet another embodiment, the substituted ammonium is a tertiary orquaternary ammonium including, without any limitation,trimethylammonium, triethylammonium, tributylammonium,diethylmethylammonium, diisopropylethylammonium, triisopropylammonium,N-methylmorpholinium, N-hydroxyethylpiperidinium, N-methylpyrrolidinium,and N, N′-dimethylpiperazinium, tetramethylammonium, tetraethylammonium,and tetrabutylammonium. These substituted ammonium compounds aregenerally commercially available in the form of various salts, or arereadily prepared from their corresponding amines by neutralization withacids.

In yet another embodiment, the substituted ammonium compound accordingto the invention is a globally cationic compound, that is, under theconditions of the entity encapsulation, typically, in aqueous solutionat a pH between about pH 2 and about pH 8, bears net positive charge,e.g. as a result of ionization (protonation) of the nitrogen atom.

In yet another embodiment, the substituted primary, secondary, ortertiary ammonium compound encapsulated into liposomes has a negativelogarithm of the acidic (deprotonation) dissociation constant (pKa) ofat least about 8.0, at least about 8.5, at least about 9.0, at least9.5, or at least about 10.0, as determined in a diluted aqueous solutionat ambient temperature (typically 25° C.). Parameter pKa is a well knowncharacteristic of ammonium compounds that generally characterizes thestrength of their basic properties, and methods for pKa determinationare conventional and routine in the art. The pKa values for many aminesand their protonated forms (ammoniums) are tabulated in reference booksof chemistry and pharmacology. See, for example, IUPAC Handbook ofPharmaceutical Salts, ed. by P. H. Stahl and C. G Wermuth, Wiley-VCH,2002; CRC Handbook of Chemistry and Physics, 82nd Edition, ed. by D. R.Lide, CRC Press, Florida, 2001, p. 8-44 to 8-56. Generally, higher pKacharacterizes stronger bases. Exemplary substituted ammonium compounds,as well as unsubstituted ammonium (listed as their conjugated aminebases) have the following pKa values: pyrrolidine,11.31;piperidine,11.12; diisopropylamine,11.05; diethylamine,10.93;triethylamime,10.75; dimethylamine,10.73; tert-butylamine,10.68;cyclohexylamine,10.66; methylamine,10.66; ethylamine,10.65;propylamine,10.54; Isopropylamine,10.53; N-ethylpiperidine,10.45;dicyclohexylamine,10.4; N-methylpiperidine,10.38;diethylmethylamine,10.35; dimethylpropylamine,10.15; trimethylamine,9.8; piperazine, 9.73 (I), 5.33 (II); 2-amino-2-methylpropanol, 9.69;N,N′-dimethylpiperazine, 9.66 (I),5.2 (II);diethyl-(2-hydroxyethyl)amine, 9.58; ethanolamine, 9.5;N-hyrdoxyethylpyrrolidine, 9.44; diethanolamine, 9.28; ammonia, 9.27;dimethyl-(2-hydroxyethyl)amine, 8.83; 2-amino-2-methylpropanediol-1,3,8.8; morpholine, 8.5; tris-(hydroxymethyl)-aminomethane, 8.3;N-methylglucamine, 8.03; triethanolamine, 7.76; N-ethylmorpholine, 7.67;N-hydroxyethylmorpholine, 7.39; imidazole, 7.03; pyridine, 5.23. As arule, substitution of alkyl or cycloalkyl group for a hydrogen in anammonium compound increases pKa value. Notably, multiple hydroxyl orether functions in the substituting alkyl groups, or the presence ofaromaticity in a nitrogen-containing heterocyclic group reduce pKa valuerelative to similar substituted ammonia without hydroxyl or etherfunctions. The compounds with more than one ammonium group usually havepKa of the second and subsequent ammonium group much lower than of thefirst one. We unexpectedly discovered that substituted ammonia withhigher pKa values, that is, formed by more strongly basic amines, weremore effective than those formed from weaker amines in stabilizing thedrug inside liposomes. For example, both IHP and SOS salts oftriethylammonium (pKa=10.75) were notably more effective thancorresponding salts of triethanolammonium (pKa=7.76) in stabilizingirinotecan within the liposomes in vivo (Example 73).

The substituted ammonium contained in the liposome composition of thepresent invention can be in any suitable form, e.g., salt. Suitablesalts include pharmaceutically acceptable salts. See, for example, P. H.Stahl, C. G. Wermuth (eds), Handbook of Pharmaceutical Salts, Wiley-VCH,Weinheim, 2002. In one embodiment, the substituted ammonium is a saltcontaining one or more polyanions of the present invention. Optimallythe counter-ion (anion) in the substituted ammonium salt of the presentinvention renders the salt water soluble, is pharmaceutically inert,capable of forming precipitates or gels when in contact with atherapeutic or detectable entity, and/or is less permeable through theliposome membrane than the substituted ammonium or its non-dissociatedamine form. In general, the substituted ammonium salt of the presentinvention forms a true solution in the intraliposomal, e.g. aqueous,space, and does not form a significant amount of a condensed phase suchas micelle, bilayer, gel, or crystalline phase. The relative amount of asubstituted ammonium and a salt-forming anion, e.g., polyanion, is at ornear the point of stiochiometric equivalency, and typically has the pHon the range of 3-9, more often, pH 4-8, dependent, for example, on thedissociation constant of the conjugated base of the substituted ammoniumion.

In general, the substituted ammonium is contained inside, that is, inthe inner (interior) space of the liposomes of the present invention. Inone embodiment, the substituted ammonium is partially or substantiallycompletely removed from the outer medium surrounding the liposomes. Suchremoval can be accomplished by any suitable means known to one skilledin the art, e.g., dilution, ion exchange chromatography, size exclusionchromatography, dialysis, ultrafiltration, precipitation, etc.

According to another feature of the present invention, it provides acomposition of liposomes containing a polyanion. The polyanion of thepresent invention can be any suitable chemical entity with more than onenegatively charged groups resulting in net negative ionic charge of morethan two units within the liposome interior, e.g., aqueous, space. Thepolyanion of the present invention can be a divalent anion, a trivalentanion, a polyvalent anion, a polymeric polyvalent anion, a polyanionizedpolyol, or a polyanionized sugar. Sulfate, phosphate, pyrophosphate,tartrate, succinate, maleate, borate, and citrate are, withoutlimitation, the examples of such di- and trivalent anions. In onepreferred embodiment, the polyanion of the present invention is apolyanionic polymer, having an organic (carbon) or inorganic backbone,and a plurality of anionic functional groups, i.e functional groupsionizable to a negative charge in a neutral aqueous solution, andintegrated or appended to the backbone. A polymer is a natural orsynthetic compound, usually of high molecular weight, consisting ofrepeated linked units, each a relatively light and simple molecule.Exemplary polyanionic polymers are polyphosphate, polyvinyl sulfate,polyvinylsulfonate, anionized polyacrylic polymers, anionized, e.g.,polysulfonated polyamines, such as polysulfonated poly(ethylene imine);polysulfated, polycarboxylated, or polyphosphorylated polysaccharides;acidic polyaminoacids; polynucleotides; other polyphosphorylated,polysulfated, polysulfonated, polyborated, or polycarboxylated polymers.Such polyvalent anions and polymers are well known in the art and manyare commercially available. A polymeric anion of the present inventionis preferably a biodegradable one, that is, capable of breaking down tonon-toxic units within the living organism. Exemplary biodegradablepolymeric anion is polyphosphate.

In another preferred embodiment, the polyanion is a polyanionized polyolor a polyanionized sugar. A polyol is an organic molecule having aplurality of hydroxyl groups linked to, e.g., linear, branched, orcyclic, carbon backbone. Thus, a polyol can be characterized in otherterms as a polyhydroxylated compound. Preferably, a majority of carbonatoms in a polyol are hydroxylated. Polyols (polyatomic alcohols) aremolecules well known in the art. Both straight chain (linear orbranched) and cyclic polyols can be used. Exemplary polyols of thepresent invention are, without limitation: ethyleneglycol; glycerol,treitol, erythritol, pentaerythritol, mannitol, glucitol, sorbitol,sorbitan, xylitol, lactitol, maltitol, fructitol, and inositol. A sugarusually comprises a cyclic acetal, a cyclic ketal, a ketone, or analdehyde group, or an adduct thereof, within a group of interlinkedpredominantly hydroxylated carbon atoms. Sugars are often naturallyoccurring compounds. Hydrolysis of sugars in aqueous medium leads tounits called monosaccharides. Typically, in an aqueous solution amonosaccharide sugar molecule of five or six carbon atoms forms a cyclichemiacetal, a ring structure. Preferably, sugars of the presentinventions are monosaccharides or disaccharides, that is, consist of oneor two monosaccharide units, each having from three to seven, preferablyfrom three to six carbon atoms. Exemplary sugars of the presentinvention are, without limitation, monosacharide hexoses, such asglucose (dextrose), galactose, mannose, fructose; monosaccharidepentoses, such as xylose, ribose, arabinose, and disaccharides, such aslactose, trehalose, sucrose, maltose, and cellobiose. Compoundscomprised of several interlinked sugar units forming a ring(cyclodextrins) and their derivatives can be also used. Reduction ofsugars is one method to obtain polyols. More stable “non-reducing” andnon-metabolizable disaccharides, such as sucrose or trehalose, arepreferred. Various polyols, monosaccharides, and disaccharides arecommercially available.

A polyanionized polyol or sugar is a polyol or a sugar having itshydroxyl groups completely or partially modified or replaced withanionic groups (anionized). Thus, a polyanionized polyol orpolyanionized sugar comprises a polyol moiety or a sugar moiety alongwith anionic groups linked thereto. Exemplary anionic groups include,without any limitation, carboxylate, carbonate, thiocarbonate,dithiocarbonate, phosphate, phosphonate, sulfate, sulfonate, nitrate,and borate. It is preferred that at least one anionic group of apolyanionized sugar or polyol is strongly anionic group, that is, ismore than 50% ionized in the broad range of pH, e.g., pH 3-12,preferably, pH 2-12, when in the aqueous medium, or, alternatively, hasa log dissociation constant (pK_(a)) of 3 or less, preferably of 2 orless. Polyanionization of a polyol or a sugar can be achieved by avariety of chemical processes well known in the art. For example,reaction of polyols and/or sugars with sulfur trioxide or chlorosulfonicacid in pyridine or 2-picoline results in some or all hydroxyl groupsesterified with sulfuric acid residues (sulfated), providing for apolysulfated sugar or polyol. Exemplary sulfated sugar of the presentinvention is sulfated sucrose including, without limitation, sucrosehexasulfate, sucrose heptasulfate, and sucrose octasulfate (See Ochi.K., et al., 1980, Chem. Pharm. Bull., v. 28, p. 638-641). Similarly,reaction with phosphorus oxychloride or diethylchlorophosphate in thepresence of base catalyst results in polyphosphorylated polyols orsugars. Polyphosphorylated polyols are also isolated from naturalsources. For example, inositol polyphosphates, such as inositolhexaphosphate (phytic acid) is isolated from corn. A variety ofsulfated, sulfonated, and phosphorylated sugars and polyols suitable topractice the present invention are disclosed, e.g., in U.S. Pat. Nos.5,783,568 and 5,281,237, which are incorporated herein by reference. Itwas unexpectedly discovered that polyanionised polyhydroxylatedcompounds with only strong acid dissociation steps, e.g. the groupshaving pKa of less than about 3.0, preferably less than about 2.0, suchas, for example, sulfate monoesters (pKa 1.0 or less), provide liposomalencapsulation with better drug retention than polyanionizedpolyhydroxylated compounds having also weakly acidic dissociation steps,such as phosphate monoesters (step 1, pKa about 1.5; step 2, pKa about6.7; see Stahl and Wermuth, Op. cit., 2002). Example 73 belowillustrates this discovery. Complexation of polyols and/or sugars withmore than one molecule of boric acid also results in a polyanionized(polyborated) product. Reaction of polyols and/or sugars with carbondisulfide in the presence of alkali results in polyanionized(polydithiocarbonated, polyxanthogenate) derivatives. A polyanionizedpolyol or sugar derivative can be isolated in the form of a free acidand neutralized with a suitable base, for example, with an alkali metalhydroxide, ammonium hydroxide, or preferably with a substituted amine,e.g., amine corresponding to a substituted ammonium of the presentinvention, in a neat form or in the form of a substituted ammoniumhydroxide providing for a polyanionic salt of a substituted ammonium ofthe present invention. Alternatively, a sodium, potassium, calcium,barium, or magnesium salt of a polyanionized polyol/sugar can beisolated and converted into a suitable form, e.g., a substitutedammonium salt form, by any known method, for example, by ion exchange.

The polyanion of the present invention usually has a charge density ofat least two, three, or four negatively charged groups per unit, e.g.,per carbon atom or ring in a carbon chain or per monosaccharide unit ina sugar. The polyanionized sugar or cyclic polyol of the presentinvention preferably has at least 75% of available hydroxyl groupspolyanionized, and more preferably 100% of available hydroxyl groupspolyanionized. In addition, polyanionization inside the liposomes of thepresent invention is usually at a level that is compatible with orfacilitates the delivery and release of the entity entrapped inside theliposomes at the site of its intended action, but decreases the releaseof the entrapped entity prematurely, i.e., before the liposome reachesits site of intended action.

According to the present invention, the degree of polyanionizationinside the liposomes can be used to regulate the releasecharacteristics, e.g., release rate and kinetics of an entity entrappedinside the liposomes. In general, the degree of polyanionization can beassessed based on the amount of polyanionized sugar or polyol relativeto the total amount of anion(s) or in the case of polyanion being theonly kind of anion, the percentage of polyanionization with respect tothe total polyanionization capacity of the polyanion, e.g.,polyanionized sugar or polyol or a mixture thereof inside the liposomesof the present invention. In one embodiment, polyanionized sugar orpolyol is mixed with one or more of other anions and the less the amountof polyanionized sugar or polyol over the amount of other anion(s), thefaster the entity is released from the liposomes.

Usually if an entrapped entity is released from the liposomes at thesite of its intended activity too slowly, the desired entity releaserate can be achieved by using a mixture of polyanionized sugar or polyolwith one or more other monovalent or polyvalent anions, e.g., chloride,sulfate, phosphate, etc. Alternatively, one can use mixtures ofpolyanionized sugar or polyols with various degrees of polyanionization.In one embodiment, the degree of polyanionization inside the liposomesof the present invention is between 0.1% to 99%, 10% to 90%, or 20% to80% of the total anion(s) inside the liposomes, e.g., with an entrappedentity.

In general, the liposome composition of the present invention cancontain one or more polyanions of the present invention in any suitableform, e.g., in the form of an acid or a salt comprising a polyanion anda cation. The amount of polyanion, e.g., polyanionized sugar or polyolcan be stoichiometrically equivalent to or different from the amount ofthe cation. In one embodiment, the liposome composition of the presentinvention contains one or more polyanion salts of a cation, whereinthere is a cation concentration gradient or a pH gradient present acrossthe liposome membrane. In another embodiment, the liposome compositionof the present invention contains one or more substituted ammoniumpolyanion salts of the present invention. In yet another embodiment, theliposome composition of the present invention contains the polyanioninside the liposomes while the polyanion in the medium containing theliposomes is partially or substantially removed by any suitable meansknown to one skilled in the art, e.g., dilution, ion exchangechromatography, size exclusion chromatography, dialysis,ultrafiltration, absorption, precipitation, etc. In still anotherembodiment, the liposome with entrapped polyanion, e.g., polyanionizedpolyol or polyanionized sugar, has also a transmembrane gradienteffective in retaining substances within the liposome. Examples of suchtransmembrane gradients are pH gradient, electrochemical potentialgradient, ammonium ion gradient, substituted ammonium ion gradient, orsolubility gradient. A substituted ammonium gradient typically includesa substituted form of ammonium ion comprising at least one C—N bond,such as, primary, quaternary, tertiary, or quaternary ammonium. Methodsof creating transmembrane gradients are routine in the art of liposomes.

According to yet another feature of the present invention, the liposomecomposition of the present invention contains one or more substitutedammoniums and/or polyanions of the present invention and a chemical orbiological entity, e.g., therapeutics or detectable entity. For example,the entity contained in the liposome composition of the presentinvention can be a therapeutic agent, ink, dye, magnetic compound,fertilizer, lure, biocatalyst, taste or odor modifying substance,bleach, or any entity that is detectable by any suitable means known inthe art, e.g., magnetic resonance imaging (MRI), optical imaging,fluorescent/luminescent imaging, or nuclear imaging techniques.Conveniently, an entity contained in or loadable to the liposomecomposition of the present invention is a weakly basic andmembrane-permeable (lipophilic) entity, e.g., an amine-containing ornitrogen base entity.

In one embodiment, the entity contained in the liposome composition ofthe present invention is a therapeutic agent.

In another embodiment, the entity contained in the liposome compositionis an anticancer entity. A partial listing of some of the commonly knowncommercially approved (or in active development) antineoplastic agentsby classification is as follows.

Structure-Based Classes: Fluoropyrimidines—5-FU, Fluorodeoxyuridine,Ftorafur, 5′-deoxyfluorouridine, UFT, S-1 Capecitabine; pyrimidineNucleosides—Deoxycytidine, Cytosine Arabinoside, 5-Azacytosine,Gemcitabine, 5-Azacytosine-Arabinoside; . Purines—6-Mercaptopurine,Thioguanine, Azathioprine, Allopurinol, Cladribine, Fludarabine,Pentostatin, 2-Chloro Adenosine; Platinum Analogues—Cisplatin,Carboplatin, Oxaliplatin, Tetraplatin, Platinum-DACH, Ormaplatin,CI-973, JM-216; Anthracyclines/Anthracenediones—Doxorubicin,Daunorubicin, Epirubicin, Idarubicin, Mitoxantrone;Epipodophyllotoxins—Etoposide, Teniposide; Camptothecins—Irinotecan,Topotecan, Lurtotecan, Silatecan, 9-Amino Camptothecin,10,11-Methylenedioxy Camptothecin, 9-Nitro Camptothecin, TAS 103,7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin,7-(2-N-isopropylamino)ethyl)-20(S)-camptothecin; Hormones and HormonalAnalogues—Diethylstilbestrol, Tamoxifen, Toremefine, Tolmudex, Thymitaq,Flutamide, Bicalutamide, Finasteride, Estradiol, Trioxifene,Droloxifene, Medroxyprogesterone Acetate, Megesterol Acetate,Aminoglutethimide, Testolactone and others; Enzymes, Proteins andAntibodies—Asparaginase, Interleukins, Interferons, Leuprolide,Pegaspargase, and others; Vinca Alkaloids—Vincristine, Vinblastine,Vinorelbine, Vindesine; Taxanes—Paclitaxel, Docetaxel.

Mechanism-Based Classes: Antihormonals—See classification for Hormonesand Hormonal Analogues, Anastrozole; Antifolates—Methotrexate,Aminopterin, Trimetrexate, Trimethoprim, Pyritrexim, Pyrimethamine,Edatrexate, MDAM; Antimicrotubule Agents—Taxanes and Vinca Alkaloids;Alkylating Agents (Classical and Non-Classical)—Nitrogen Mustards(Mechlorethamine, Chlorambucil, Melphalan, Uracil Mustard),Oxazaphosphorines (Ifosfamide, Cyclophosphamide, Perfosfamide,Trophosphamide), Alkylsulfonates (Busulfan), Nitrosoureas (Carmustine,Lomustine, Streptozocin), Thiotepa, Dacarbazine and others;Antimetabolites—Purines, pyrimidines and nucleosides, listed above;Antibiotics—Anthracyclines/Anthracenediones, Bleomycin, Dactinomycin,Mitomycin, Plicamycin, Pentostatin, Streptozocin; topoisomeraseInhibitors—Camptothecins (Topo I), Epipodophyllotoxins, m-AMSA,Ellipticines (Topo II); Antivirals—AZT, Zalcitabine, Gemcitabine,Didanosine, and others; Miscellaneous Cytotoxic Agents—Hydroxyurea,Mitotane, Fusion Toxins, PZA, Bryostatin, Retinoids, Butyric Acid andderivatives, Pentosan, Fumagillin, and others.

In addition to the above, an anticancer entity include without anylimitation, any topoisomerase inhibitor, vinca alkaloid, e.g.,vincristine, vinblastine, vinorelbine, vinflunine, and vinpocetine,microtubule depolymerizing or destabilizing agent, microtubulestabilizing agent, e.g., taxane, aminoalkyl or aminoacyl analog ofpaclitaxel or docetaxel, e.g.,2′-[3-(N,N-Diethylamino)propionyl]paclitaxel,7-(N,N-Dimethylglycyl)paclitaxel, and 7-L-alanylpaclitaxel, alkylatingagent, receptor-binding agent, tyrosine kinase inhibitor, phosphataseinhibitor, cycline dependent kinase inhibitor, enzyme inhibitor, aurorakinase inhibitor, nucleotide, polynicleotide, and farnesyltransferaseinhibitor.

In another embodiment, the entity contained in the liposome compositionof the present invention is a therapeutic agent of anthracyclinecompounds or derivatives, camptothecine compounds or derivatives,ellipticine compounds or derivatives, vinca alkaloinds or derivatives,wortmannin, its analogs and derivatives, or pyrazolopyrimidine compoundswith the aurora kinase inhibiting properties.

In yet another embodiment, the entity contained in the liposomecomposition of the present invention is an anthracycline drug,doxorubicin, daunorubicin, mitomycin C, epirubicin, pirarubicin,rubidomycin, carcinomycin, N-acetyladriamycin, rubidazone,5-imidodaunomycin, N-acetyldaunomycine, daunoryline, mitoxanthrone; acamptothecin compound, camptothecin, 9-aminocamptothecin,7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin,10,11-methylenedioxycamptothecin,9-amino-10,11-methylenedioxycamptothecin,9-chloro-10,11-methylenedioxycamptothecin, irinotecan, topotecan,lurtotecan, silatecan,(7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin,7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin,7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin; an ellipticinecompound, ellipticine, 6-3-aminopropyl-ellipticine,2-diethylaminoethyl-ellipticinium and salts thereof, datelliptium,retelliptine.

In yet another embodiment, the entity contained in the liposome of thepresent invention is a pharmaceutical entity including, withoutlimitation any of the following: antihistamine ethylenediaminederivatives (bromphenifamine, diphenhydramine); Anti-protozoal:quinolones (iodoquinol); amidines (pentamidine); antihelmintics(pyrantel); anti-schistosomal drugs (oxaminiquine); antifungal triazolederivatives (fliconazole, itraconazole, ketoconazole, miconazole);antimicrobial cephalosporins (cefazolin, cefonicid, cefotaxime,ceftazimide, cefuoxime); antimicrobial beta-lactam derivatives(aztreopam, cefmetazole, cefoxitin); antimicrobials of erythromycinegroup (erythromycin, azithromycin, clarithromycin, oleandomycin);penicillins (benzylpenicillin, phenoxymethylpenicillin, cloxacillin,methicillin, nafcillin, oxacillin, carbenicillin); tetracyclines; otherantimicrobial antibiotics, novobiocin, spectinomycin, vancomycin;antimycobacterial drugs: aminosalicycic acid, capreomycin, ethambutol,isoniazid, pyrazinamide, rifabutin, rifampin, clofazime; antiviraladamantanes: amantadine, rimantadine; quinidine derivatives:chloroquine, hydroxychloroquine, promaquine, qionone; antimicrobialqionolones: ciprofloxacin, enoxacin, lomefloxacin, nalidixic acid,norfloxacin, ofloxacin; sulfonamides; urinary tract antimicrobials:methenamine, nitrofurantoin, trimetoprim; nitroimidazoles:metronidazole; cholinergic quaternary ammonium compounds (ambethinium,neostigmine, physostigmine); anti-Alzheimer aminoacridines (tacrine);anti-Parkinsonal drugs (benztropine, biperiden, procyclidine,trihexylhenidyl); anti-muscarinic agents (atropine, hyoscyamine,scopolamine, propantheline); adrenergic dopamines (albuterol,dobutamine, ephedrine, epinephrine, norepinephrine, isoproterenol,metaproperenol, salmetrol, terbutaline); ergotamine derivatives;myorelaxants or curane series; central action myorelaxants; baclophen,cyclobenzepine, dentrolene; nicotine; beta-adrenoblockers (acebutil,amiodarone); benzodiazepines (ditiazem); antiarrhythmic drugs(diisopyramide, encaidine, local anesthetic series—procaine,procainamide, lidocaine, flecaimide), quinidine; ACE inhibitors:captopril, enelaprilat, fosinoprol, quinapril, ramipril; antilipidemics:fluvastatin, gemfibrosil, HMG-coA inhibitors (pravastatin); hypotensivedrugs: clonidine, guanabenz, prazocin, guanethidine, granadril,hydralazine; and non-coronary vasodilators: dipyridamole.

According to the present invention, the entity contained in the liposomecomposition of the present invention can also be a pre-entity, e.g., apro-drug or an agent that is capable of being converted to a desiredentity upon one or more conversion steps under a condition such as achange in pH or an enzymatic cleavage of a labile bond. Such conversionmay occur after the release of the pro-drug from the liposome interiorat the intended site of the drug/liposome action. However, thepre-entity can be converted into the desired active entity inside theliposomes of the present invention prior to the use of the liposomes asa delivery vehicle, e.g., administration to a patient. For example, anentity can be modified into a pre-entity so that it is easier to beloaded into the liposomes and then it can be converted back into thedesired entity once it is inside the liposomes of the present invention.In this manner, according to the present invention, the entities thatare generally not amenable to “active”, “remote” or other gradient-basedloading methods, can be effectively loaded into liposomes, e.g., intothe liposome interior space, in their native, unmodified form.

Globally cationic compounds, that is, compounds capable of attaining anet positive ionic charge under the liposome loading conditions,especially the compounds containing a titratable amine, are known toeffectively load into liposomes exhibiting transmembrane ion gradients.If an entity of interest is an organic compound and is not not aglobally cationic compound having a titratable amine, a derivativethereof having the requisite ionic properties can be prepared by asuitable modification, e.g., according to the methods described inWoodle et al., in WO 96/25147. For example, an amine group can beintroduced by esterification of a hydroxyl group of the entity with anamino acid. Alternatively, a hydrophobic group can be introduced into awater-soluble compound to aid in its partition into the liposomemembrane and subsequent traversing of the membrane to the intraliposomalcompartment, i.e., inside the liposomes. Another useful modification tocreate a liposome-loadable pre-entity is the formation of a carbonylgroup adduct, e.g., a hydrazone, an oxime, an acetal, or a ketal. Amodified amino-containing group can be hydrolyzed or otherwisechemically split from the modified compound after the loading of themodified compound into the liposomes according to the present invention.Typical processes to intraliposomally regenerate the entity from apre-entity are hydrolysis, photolysis, radiolysis, thiolysis,ammonolysis, reduction, substitution, oxidation, or elimination. Theseprocesses can be effected, without limitation, by the change of pH or byan enzymatic action. For example, paclitaxel or docetaxel, a non-ionicentities, are converted into their 2′-(diethylaminopropionyl)- or7′-(diethylaminopropionyl) esters, which are weak bases (pre-entities).After loading into the liposomes by any known method, including, withoutlimitation, “active”, “remote”, “transmembrane-gradient-based” or“solubility gradient based” methods, and/or the methods of the presentinvention, the intraliposomal 2′-(diethylaminopropionyl)-paclitaxel isconverted into original paclitaxel by stimulating its hydrolysis throughthe increase of pH to above pH 7.0. Thus, a liposome encapsulating aneutral taxane molecule within its interior space is obtained with thedrug/lipid ratio of over 0.05 mole per mole of the liposome lipid,without the help of hydrophilic covalent modifications of the taxanemolecule (e.g. by attachment of PEG), cyclodextrine taxane compexes, ortaxane-solubilizing, micelle-forming surfactants.

According to the present invention, the liposomes contained in theliposome composition of the present invention can be any liposome knownor later discovered in the art. In general, the liposomes of the presentinvention can have any liposome structure, e.g., structures having aninner space sequestered from the outer medium by one or more lipidbilayers, or any microcapsule that has a semi-permeable membrane with alipophilic central part where the membrane sequesters an interior. Alipid bilayer can be any arrangement of amphiphilic moleculescharacterized by a hydrophilic part (hydrophilic moiety) and ahydrophobic part (hydrophobic moiety). Usually amphiphilic molecules ina bilayer are arranged into two dimensional sheets in which hydrophobicmoieties are oriented inward the sheet while hydrophilic moieties areoriented outward. Amphiphilic molecules forming the liposomes of thepresent invention can be any known or later discovered amphiphilicmolecules, e.g., lipids of synthetic or natural origin or biocompatiblelipids. Liposomes of the present invention can also be formed byamphiphilic polymers and surfactants, e.g., polymerosomes and niosomes.For the purpose of this disclosure, without limitation, theseliposome-forming materials also are referred to as “lipids”.

According to the present invention, the liposomes contained in theliposome composition of the present invention can also be targetingliposomes, e.g., liposomes containing one or more targeting moieties orbiodistribution modifiers on the surface of the liposomes. A targetingmoiety can be any agent that is capable of specifically binding orinteracting with a desired target. In one embodiment, a targeting moietyis a ligand. The ligand, according to the present invention,preferentially binds to and/or internalizes into, a cell in which theliposome-entrapped entity exerts its desired effect (a target cell). Aligand is usually a member of a binding pair where the second member ispresent on or in a target cells or in a tissue comprising the targetcell. Examples of ligands suitable for the present invention are: thefolic acid, protein, e.g., transferrin, growth factor, enzyme, peptide,receptor, antibody or antibody fragment, such as Fab′, Fv, single chainFv, single-domain antibody, or any other polypeptide comprisingantigen-binding sequences (CDRs) of an antibody molecule. Aligand-targeted liposome wherein a targeting moiety is an antibody or atarget antigen-binding fragment thereof is called an immunoliposome. Ina preferred embodiment, the liposome carrying a targeting moiety, e.g.,a ligand, is internalized by a target cell. In yet another embodiment, atargeting moiety is a ligand that specifically interacts with a tyrosinekinase receptor such as, for example, EGFR, HER2, HER3, HER4, PD-GFR,VEGFR, bFGFRor IGFR receptors. In still another embodiment, thetargeting moiety specifically interacts with a growth factor receptor,an angiogenic factor receptor, a transferrin receptor, a cell adhesionmolecule, or a vitamin receptor.

According to another embodiment of the present invention, the liposomescontained in the liposome composition exhibit a transmembraneconcentration gradient of a substituted ammonium and/or polyanion of thepresent invention. Preferably, the higher concentration is in theinterior (inner) space of the liposomes. In addition, the liposomecomposition of the present invention can include one or moretrans-membrane gradients in addition to the gradient created by thesubstituted ammonium and/or polyanion of the present invention. Forexample, the liposomes contained in the liposome composition of thepresent invention can additionally include a transmembrane pH gradient,ion gradient, electrochemical potential gradient, and/or solubilitygradient.

According to yet another embodiment of the present invention, theliposome composition of the present invention can be provided in a kitcomprising a container with the liposomes, and optionally, a containerwith the entity and an instruction, e.g., procedures or informationrelated to using the liposome composition in one or more applications.Such instruction can be provided via any medium, e.g., hard paper copy,electronic medium, or access to a database or website containing theinstruction.

The liposome membrane composition of the present invention can be madeby any suitable method known to or later discovered by one skilled inthe art. In general, a variety of lipid components can be used to makethe liposomes of the present invention. Lipid components usuallyinclude, but are not limited to (1) uncharged lipid components, e.g.,cholesterol, ceramide, diacylglycerol, acyl(poly ethers) oralkylpoly(ethers); (2) neutral phospholipids, e.g.,diacylphosphatidylcholines, sphingomyelins, anddiacylphosphatidylethanolamines, (3) anionic lipids, e.g.,diacylphosphatidylserine, diacylphosphatidylglycerol,diacylphosphatidate, cardiolipin, diacylphosphatidylinositol,diacylglycerolhemisuccinate, diaclyglycerolhemigluratate,cholesterylhemisuccinate, cholesterylhemiglutarate, and the like; (4)polymer-conjugated lipids, e.g., N-[methoxy-(poly(ethyleneglycol)diacylphosphatidylethanolamine, poly(ethyleneglycol)-diacylglycerol, poly(ethylene glycol)-ceramide; and (5) cationiclipids, e.g., 1,2,-diacyl-3-trimethylammonium-propane (DOTAP),dimethyldioctadecylammonium bromide (DDAB), and1,2-diacyl-sn-glycero-3-ethylphosphocholine. Monoacyl-substitutedderivatives of these lipids, as well as di- and monoalkyl-analogs can bealso employed.

Various lipid components can be selected to fulfill, modify or impartone or more desired functions. For example, phospholipid can be used asprincipal vesicle-forming lipid. Inclusion of cholesterol is useful formaintaining membrane rigidity and decreasing drug leakage.Polymer-conjugated lipids can be used in the liposomal formulation toincrease the lifetime of circulation via reducing liposome clearance byliver and spleen, or to improve the stability of liposomes againstaggregation during storage, in the absence of circulation extendingeffect. While inclusion of PEG-lipids in the amount 1 mol % or above ofthe liposome lipid is asserted to have a several-fold prolongation ofthe liposome blood circulation time (see, e.g., U.S. Pat. No.5,013,556), we have surprisingly discovered that liposomes of thepresent invention are quite long-circulating, and the addition ofPEG-lipid to the liposome composition only extended the circulationlongevity for less than two-fold, if at all. In addition,charge-modulating (titratable) lipids can be used to help delivery ofliposome encapsulated entities to cytosolic or nuclear targets viafacilitating some classes of entities escaping the confines of endosomalpathway.

In one embodiment, the liposomes of the present invention includelecithin, cholesterol, and an amphipathic polymer. The lecithin includedin the liposomes of the present invention can be a natural lecithin, ahydrogenated natural lecithin, a synthetic lecithin,1,2-distearoyl-lecithin, dipalmitoyl lecithin, dimyristoyl lecithin,dioleolyl lecithin, 1-stearoyl-2-oleoyl lecithin, or1-palmitoyl-2-oleoyl lecithin whereas the amphipathic polymer can be apolyethylene glycol-lipid derivative, e.g., polyethylene glycolphosphatidylethanolamine, polyethylene glycol-diacylglycerol, orpolyethyleneglycol-ceramide derivative, where the poly(ethylene glycol)portion has molecular weight from about 250 to about 20,000, mostcommonly from about 500 to about 5,000. In another embodiment, thelecithin and cholesterol ratio in the liposomes of the present inventionis about 3:2 by mole. In yet another embodiment, the amphipathic polymeris at least 0.1 mole % of the liposome-forming lipid in the liposomes ofthe present invention. In yet another embodiment, the amount of anamphipathic polymer is between 0.1 mole % and 1 mole % of theliposome-forming lipid in the liposomes of the present invention.Preferably, the amphipathic polymer is a neutral polymer, i.e. possessesunder the drug loading conditions the net ionic charge of zero, forexample, PEG-diacylglycerol, PEG-dialkylglycerol, or PEG-ceramide. Itwas unexpectedly discovered that inclusion of ionically neutralamphipathic lipids up to PEG-lipid content of about 5.7 mol. % of totallipid afford high efficiency liposome loading of, e.g., vinca alkaloids,such as vinorelbine, while in the case of anionically charged PEG-DSPEthe loading efficiency noticeably declined at the PEG-lipid content of1.6 mol. % or more (Example 72).

In still another embodiment, the liposomes of the present inventioncontain a camptothecin derivative, e.g., a camptothecin prodrug such asirinotecan and is comprised of lecithin and cholesterol, e.g., at aratio of about 3:2 by mole, and an amphipathic polymer, e.g., at anamount of at least 0.1 mole % or less than 1% of the liposome-forminglipid.

Liposomes of the present invention can be made by any method that isknown or will become known in the art. See, for example, G. Gregoriadis(editor), Liposome Technology, vol. 1-3, 1st edition, 1983; 2nd edition,1993, CRC Press, Boca Raton, Fla. Examples of methods suitable formaking liposome composition of the present invention include extrusion,reverse phase evaporation, sonication, solvent (e.g., ethanol)injection, microfluidization, detergent dialysis, ether injection, anddehydration/rehydration. The size of liposomes can be controlled bycontrolling the pore size of membranes used for low pressure extrusionsor the pressure and number of passes utilized in microfluidisation orany other suitable methods. In one embodiment, the desired lipids arefirst hydrated by thin-film hydration or by ethanol injection andsubsequently sized by extrusion through membranes of a defined poresize; most commonly 0.05 μm, 0.08 μm, or 0.1 μm.

Liposome compositions containing the substituted ammonium and/orpolyanion of the present invention inside the liposomes can be made byany suitable methods, e.g., formation of liposomes in the presence ofthe substituted ammonium and/or polyanion of the present invention,e.g., in the form of salt. The substituted ammonium and/or polyanionoutside of the liposomes can be removed or diluted either followingliposome formation or before loading or entrapping a desired entity.Alternatively, liposome composition containing the substituted ammoniumand/or polyanion of the present invention can be made via ion exchangemethod directly or via an intermediate free acid step having a gradientof substituted ammonium of the present invention, e.g., substitutedammonium salt of polyanionized sugar or polyol. Such liposomes can beneutralized using the amine or its salt with a volatile acid, e.g.,carbonate. The resulting liposome solution can be used directly oralternatively the salt contained therein can be removed if desired,e.g., by evaporation and crystallization followed by dissolution in anaqueous medium.

Preferably, the liposome composition of the present invention has atransmembrane concentration gradient of the substituted ammonium and/orpolyanion, e.g., the concentration of the substituted ammonium and/orpolyanion salt inside the liposome is higher, usually at least 100 timeshigher, than the concentration of the substituted ammonium and/orpolyanion in the medium outside the liposome.

In one embodiment, the concentration of the substituted ammonium and/orpolyanion salt inside the liposome is at least 100 times higher than theconcentration of the substituted ammonium and/or polyanion salt in themedium outside the liposome and is at least at a concentration of about10 mM, 50 mM, 0.1M, 0.2M, 0.5M, 0.6M, 0.7M, or 1.0M, wherein molarity iscalculated based on the substituted ammonium. In another embodiment, theconcentration of the substituted ammonium and/or polyanion salt insidethe liposome is at least 100 times higher than the concentration of thesubstituted ammonium and/or polyanion salt in the medium outside theliposome and is at a concentration of about 0.65M or about 1.0M.

In addition, the liposome composition of the present invention usuallyhas a pH outside which is compatible with or helpful for maintaining thestability of a desired entity during the loading process, along with thehigh loading efficiency, e.g., above 90% entrapment. For example, pH inthe range of 4-7, or pH 4.5-6.5, is preferred. In particular, accordingto the present invention, loading of a camptothecin compound, e.g.,topotecan or irinotecan, is best accomplished at the pH of the outermedium in the range between about 4.0 and about 7.0, more preferablybetween about pH 5.0 and pH 6.5. Loading of a vinca derivative, e.g.,vincristine, vinorelbine, or vinblastine is best accomplished at pHabout 5.0-7.0, more preferably at pH about 6.5.

According to the present invention, a desired entity can be loaded orentrapped into the liposomes by incubating the desired entity with theliposomes of the present invention in an aqueous medium at a suitabletemperature, e.g., a temperature above the component lipids' phasetransition temperature during loading while being reduced below thephase transition temperature after loading the entity. The incubationtime is usually based on the nature of the component lipids, the entityto be loaded into the liposomes, and the incubation temperature.Typically, the incubation times of few minutes to several hours aresufficient. Because high entrapment efficiencies of more than 85%,typically more than 90%, are achieved, there is usually no need toremove unentrapped entity. If there is such a need, however, theunentrapped entity can be removed from the composition by various mean,such as, for example, size exclusion chromatography, dialysis,ultrafiltration, adsorption, or precipitation. It was unexpectedly foundthat maintaining of the low ionic strength during the incubation of anentity, such as, in particular, a camptothecin derivative or a vincaalkaloid derivative, with the liposomes of the present invention,followed by the increase in ionic strength at the end of the incubation,results in higher loading efficiency, better removal of unentrappeddrug, and better liposome stability against aggregation. Typically, theincubation is conducted, e.g., in an aqueous solution, at the ionicstrength of less than that equivalent to 50 mM NaCl, or more preferably,less than that equivalent to 30 mM NaCl. Following the incubation, aconcentrated salt, e.g., NaCl, solution may be added to raise the ionicstrength to higher than that of 50 mM NaCl, or more preferably, higherthan that of 100 mM NaCl. Without being bound by a theory, wehypothesize that the increase of ionic strength aids dissociation of theentity from the liposome membrane, leaving substantially all entityencapsulated within the liposomal interior space.

In general, the entity-to-lipid ratio, e.g., drug load ratio obtainedupon loading an entity depends on the amount of the entity entrappedinside the liposomes, the concentration of entrapped substitutedammonium and/or polyanion, e.g., salt, the physicochemical properties ofthe entrapped entity and the type of counter-ion (anion), e.g.,polyanion used. Because of high loading efficiencies achieved in thecompositions and/or by the methods of the present invention, theentity-to-lipid ratio for the entity entrapped in the liposomes is over80%, over 90%, and typically more than 95% of the entity-to-lipid ratiocalculated on the basis of the amount of the entity and the liposomelipid taken into the loading process (the “input” ratio). Indeed,practically 100% (quantitative) encapsulation is common. The entity-tolipid ratio in the liposomes can be characterized in terms of weightratio (weight amount of the entity per weight or molar unit of theliposome lipid) or molar ratio (moles of the entity per weight or molarunit of the liposome lipid). One unit of the entity-to-lipid ratio canbe converted to other units by a routine calculation, as exemplifiedbelow. The weight ratio of an entity in the liposomes of the presentinvention is typically at least 0.05, 0.1, 0.2, 0.35, 0.5, or at least0.65 mg of the entity per mg of lipid. In terms of molar ratio, theentity-to-lipid ratio according to the present invention is at leastfrom about 0.02, to about 5, preferably at least 0.1 to about 2, andmore preferably, from about 0.15 to about 1.5 moles of the drug per moleof the liposome lipid. In one embodiment, the entity-to-lipid ratio,e.g., drug load ratio of camptothecin derivatives is at least 0.1, e.g.,0.1 mole of camptothecin derivative per one mole of liposome lipid, andpreferably at least 0.2. In another embodiment, the entity-to-lipidratio, e.g., drug load is at least about 300 mg entity (e.g., vincaalkaloid or a derivative thereof) per mg of liposome-forming lipid. Inyet another embodiment, the entity-to-lipid ratio, e.g., drug load is atleast about 500 mg entity (e.g. camptothecin derivative or camprothecinprodrug) per mg of liposome-forming lipid. Surprisingly, the inventionafforded stable and close to quantitative liposomal encapsulation of acamptothecin derivative drug, e.g., irinotecan, at the drug-to-lipidratio of over 0.8 mmol of the entity per 1 g of liposome lipid, over 1.3mmol of entity per 1 g of liposome lipid, and even at high as 1.7 mmolentity per 1 g liposome lipid (see Example 74).

If the liposome comprises a phospholipid, it is convenient to expressthe entity content in the units of weight (mass) amount of the drug permolar unit of the liposome phospholipid, e.g., mg drug/mmol ofphospholipid. However, a person skilled in the art would appreciate thatthe drug content can be equivalently expressed in a manner independentof the presence of phospholipids in a liposome, and furthermore, can beequivalently expressed in terms of a molar amount of the drug per unit(mass or molar) of the liposome lipid content. For example, a liposomecontaining 3 molar parts of distearoylphosphatidylcholine (DSPC,molecular weight 790), 2 molar parts of cholesterol (molecular weight387), and 0.015 molar parts of poly(ethylene glycol)-derivatizeddistearoylphosphatidylethanolamine (PEG-DSPE, molecular weight 2750),and containing a drug doxorubicin (molecular weight 543.5) at thedrug/lipid ratio of 150 mg/mmol phospholipid, the same drug content canbe equivalently expressed in terms of mg drug/mg total lipid as follows:

(a) Calculate the molar amounts of liposome lipid components normalizedto the molar unit of liposome phospholipids (DSPC and PEG-DSPE in thisexample) by dividing the molar quantity of a component by the total ofthe molar quantities of the liposome phospholipids:

-   -   DSPC 3/(3+0.015)=0.99502    -   Cholesterol 2/(3+0.015)=0.66335    -   PG-DSPE 0.015/(3+0.015)=0.00498        (b) Calculate the mass amount of total liposome lipid        corresponding to a unit molar amount of liposome phospholipid        and the components molecular weights:

Total lipid, mg/mmolphospholipid=0.99502×790+0.66335×387+0.00498×2750=1056.48

(c) Calculate the mass amount of drug per mass unit of total lipid bydividing the drug content expressed in mass units per molar unit ofphospholipid by the number obtained in step (b):

Doxorubicin, mg/mg total lipid=150/1056.48=0.14198.

(d) Calculate the molar amount of the drug per unit mass of total lipidby dividing the number obtained in step (c) by the drug molecular weight(in this case, 543.5):

Doxorubicin, mmol/g total lipid=0.14198/543.5×1000=0.261.

(e) Calculate the molar part of phospholipids in the liposome lipidmatrix:

Phospholipid molar part=(total moles of phospholipids)/(total molesamount of lipids)=(3+0.015)/(3+2+0.015)=0.6012.

(f) Calculate the molar ratio of doxorubicin to total lipid.

Doxorubicin, mol/mol of total lipid=(Phospholipid molarpart)×(Doxorubicin, g/mole phospholipid)/(Doxorubicin molecularweight)=0.6012×150/543.5=0.166

Thus, the relationship between drug-to-lipid and drug-to-phospholipidratio expressed in various units is readily established. As used herein,a “lipid” includes, without limitation, any membrane-forming componentsof the liposome membrane, such as, for example, polymers and/ordetergents.

The liposome entrapped substituted ammonium and/or polyanion saltsolution of the present invention usually has an osmotic strength(osmolality) which helps to keep the liposomes stable against osmoticdamage (swelling and/or burst) without sacrificing the loading capacityof the liposomes. In one embodiment, the osmolality of the liposomecomposition of the present invention is in the range of 0.1 to 1.5mol/kg or, preferably, 0.2 to 1.0 mol/kg. Surprisingly, we found thatliposomes of the present invention are stable against adverse effect ofhigh intraliposomal osmotic strength on the drug loading. Intraliposomalosmolarities of as high as 0.727 mol/kg were well tolerated, resultingin practically quantitative loading of a drug up to the theoreticalmaximum of stoichiometric exchange of intraliposomal substitutedammonium ions for molecules of the drug (in the case of irinotecan, onedrug molecule per one substituted ammonium ion), even though theosmolarity of the extraliposomal aqueous medium during the co-incubationof the drug and the liposomes was close to the physiological value ofabout 0.3 mol/kg (Example 74).

In general, the liposome composition of the present invention is quitestable during storage, e.g., as measured by the percentage of entrappedentity released outside of the liposomes or still maintained inside ofthe liposomes after a certain time period from the initial loading ofthe entity inside the liposomes of the present invention. For example,the liposome composition of the present invention is stable at 4° C. forat least 6 months, e.g., less than 10% of entrapped entity is released 6months after the initial loading of the entity. In one embodiment, theliposome composition of the present invention is stable at 4° C. for atleast 2 years, e.g., less than 20% of entrapped entity is released 2years after the initial loading of the entity.

It is advantageous for a liposome-entrapped entity to remainencapsulated in the liposome until the liposome reaches the site of itsintended action, e.g., in the case of a liposomal antitumor drugadministered in a patient, a tumor. The liposomes of the presentinvention showed surprising stability against the release (leakage) ofthe entrapped entity under in vivo conditions, e.g. in the blood of amammal. The exposure time needed for 50% release of the entrappedentity, e.g. drug, from the liposomes (half-release time) in the bloodof a rat in vivo was more than 24 hours. In particular, liposomes loadedwith vinca alkaloid drugs, e.g., vinblastine, vincristine, andvinorelbine, showed remarkable stability against drug leakage in vivo,with half-release time of at least 24 hours, or the amount of entityremaining encapsulated after 24 hours in the blood in vivo at leastabout 50% of the pre-administration value. Typically the half-releasetime over 33 hours, or the amount of encapsulated entity remainingencapsulated after 24 hours in the blood in vivo at least about 60%, wasobserved; and even the half-release time over 46 hours, or the amount ofencapsulated entity after 24 hours in the blood in vivo at least about70% of the pre-administration value, was common. Sometimes thehalf-release time for an encapsulated drug in the blood in vivo was over93 hours, and even over 120 hours. The liposome loaded with camptothecinderivatives, such as topotecan and irinotecan, also showed exceptionalin vivo stability in the blood, with 79-85% of the original drug loadremaining encapsulated after 24 hours. Remarkably, the liposomes of thepresent invention, while having such low in vivo drug release rate inthe blood circulation, showed substantial in vivo antitumor activityexceeding that of the free drug (i.e administered as a solution).

The liposomes of the present invention provided unexpected combinationof the high efficiency of the entrapped therapeutic agent and lowtoxicity. In general, the activity of a therapeutic entity liposomallyencapsulated according to the present invention, e.g., theanti-neoplastic activity of a camptothecin derivative in a mammal, is atleast equal to, at least two times higher, or at least four times higherthan the activity of the therapeutic entity if it is administered in thesame amount via its routine non-liposome formulation, e.g., withoutusing the liposome composition of the present invention, while thetoxicity of the liposomally encapsulated entity does not exceed, is atleast twice, at least three times, or at least four times lower thanthat of the same therapeutic entity administered in the same dose andschedule but in a free, non-encapsulated form. For example, it isgenerally known that liposomal encapsulation of anti-cancer camptothecinderivatives by the published methods of others results in the increasedtoxicity (lower maximum tolerated dose, lower 50% lethality dose)compared to unencapsulated drug. See U.S. Pat. Nos. 6,355,268;6,465,008; Colbern, et al. Clinical Cancer Res. 1998, v. 4, p.3077-3082; Tardi, et al. Cancer Res., 2000, v. 60, p. 3389-3393;Emerson, et al. Clinical Cancer Res. 2000, v. 6, p. 2903-2912.Liposomally encapsulated camptothecin pro-drugs, such as irinotecan(CPT-11), which is a water-soluble, cationic camptothecin pro-drugderivative, have substantially higher, e.g. at least 4 times, and even10 times, higher antitumor activity assessed in an in vivo tumor modelthan the drug in the absence of a liposomal formulation, e.g., in a free(solution) form. This is even more remarkable since a therapeuticcompound, e.g., a camptothecin pro-drug, requires enzymatic activation,e.g., by the action of endogenous non-specific carboxylesterase, butaccording to the present invention is encapsulated substantially withinthe interior space of the liposome. On the other hand, surprisingly, thetoxicity of camptothecin prodrug such as CPT-11 in the liposomal form(drug/lipid mass ratio over 0.1, e.g., 0.2-0.6 or more) according to thepresent invention was over 2 times, over 3 times, and even over 4 timeslower that than of the free (unencapsulated) pro-drug CPT-11. Moreover,a prolonged drug release from the CPT-11 liposomes in vivo was achieved,with more than 50%, and even more than 70% (79-86%) of the original drugcontent still remaining in the liposomes 24 hours after administrationinto the bloodstream, and with half-release times in excess of 24 hours,typically in excess of 48 hours. The prolonged remanence of the drug inthe liposome in vivo was associated with higher antitumor effect.Surprisingly, the slowest in vivo CPT-11 release and the highestantitumor activity was observed in the liposomes containinglow-molecular polyanionized sugar derivative (sucrose octasulfate)rather than a polymeric anion (polyphosphate) (Example 15).

According to another embodiment of the present invention, the liposomecomposition of the present invention can be provided as a pharmaceuticalcomposition containing the liposome composition of the present inventionand a carrier, e.g., pharmaceutically acceptable carrier. Examples ofpharmaceutically acceptable carries are normal saline, isotonicdextrose, isotonic sucrose, Ringer's solution, and Hanks' solution. Abuffer substance can be added to provide pH optimal for storagestability. For example, pH between about 6.0 and about 7.5, morepreferably pH about 6.5, is optimal for the stability of liposomemembrane lipids, and provides for excellent retention of the entrappedentities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES),morpholipo-ethylsulfonate (MES), succinate, tartrate, and citrate,typically at 2-20 mM concentration, are exemplary buffer substances.Other suitable carriers include, e.g., water, buffered aqueous solution,0.4% NaCl, 0.3% glycine, and the like. Protein, carbohydrate, orpolymeric stabilizers and tonicity adjusters can be added, e.g.,gelatin, albumin, dextran, or polyvinylpyrrolidone. The tonicity of thecomposition can be adjusted to the physiological level of 0.25-0.35mol/kg with glucose or a more inert compound such as lactose, sucrose,mannitol, or dextrin. These compositions may be sterilized byconventional, well known sterilization techniques, e.g., by filtration.The resulting aqueous solutions may be packaged for use or filteredunder aseptic conditions and lyophilized, the lyophilized preparationbeing combined with a sterile aqueous medium prior to administration.

The pharmaceutical liposome compositions can also contain otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alpha-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of the liposomes of the present invention in the fluidpharmaceutical formulations can vary widely, i.e., from less than about0.05% usually or at least about 2-10% to as much as 30 to 50% by weightand will be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension. Alternatively, liposome pharmaceutical compositionscomposed of irritating lipids may be diluted to low concentrations tolessen inflammation at the site of administration.

The amount of liposome pharmaceutical composition administered willdepend upon the particular therapeutic entity entrapped inside theliposomes, the disease state being treated, the type of liposomes beingused, and the judgment of the clinician. Generally the amount ofliposome pharmaceutical composition administered will be sufficient todeliver a therapeutically effective dose of the particular therapeuticentity.

The quantity of liposome pharmaceutical composition necessary to delivera therapeutically effective dose can be determined by routine in vitroand in vivo methods, common in the art of drug testing. See, forexample, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbookof Anticancer Drug Development, L W W, 2003. Therapeutically effectivedosages for various therapeutic entities are well known to those ofskill in the art; and according to the present invention a therapeuticentity delivered via the pharmaceutical liposome composition of thepresent invention provides at least the same, or 2-fold, 4-fold, or10-fold higher activity than the activity obtained by administering thesame amount of the therapeutic entity in its routine non-liposomeformulation. Typically the dosages for the liposome pharmaceuticalcomposition of the present invention range between about 0.005 and about500 mg of the therapeutic entity per kilogram of body weight, mostoften, between about 0.1 and about 100 mg therapeutic entity/kg of bodyweight.

Typically, the liposome pharmaceutical composition of the presentinvention is prepared as a topical or an injectable, either as a liquidsolution or suspension. However, solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The composition can also be formulated into an enteric-coatedtablet or gel capsule according to known methods in the art.

The liposome composition of the present invention can be administered inany way which is medically acceptable which may depend on the conditionor injury being treated. Possible administration routes includeinjections, by parenteral routes such as intramuscular, subcutaneous,intravenous, intraarterial, intraperitoneal, intraarticular,intraepidural, intrathecal, or others, as well as oral, nasal,ophthalmic, rectal, vaginal, topical, or pulmonary, e.g., by inhalation.For the delivery of liposomally drugs formulated according to theinvention, to tumors of the central nervous system, a slow, sustainedintracranial infusion of the liposomes directly into the tumor (aconvection-enhanced delivery, or CED) is of particluar advantage. SeeSaito, et al., Cancer Research, vol. 64, p. 2572-2579, 2004; Mamot, etal., J. Neuro-Oncology, vol. 68, p. 1-9, 2004._ The compositions mayalso be directly applied to tissue surfaces. Sustained release, pHdependent release, or other specific chemical or environmental conditionmediated release administration is also specifically included in theinvention, e.g., by such means as depot injections, or erodibleimplants.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used.

Example 1. Preparation of the Solutions of Substituted Ammonium Salts

Trialkylammonium and dialkylammonium sulfate solutions useful forloading drugs (e.g., doxorubicin) into liposomes were prepared bydiluting sulfuric acid with water to a concentration of 0.25 M and thentitrating the sulfuric acid solution with one of a variety of amines.The substituted amines used in this example were triethylamine,trimethylamine, dimethylamine, diethylamine, or diethanolamine. Afterthe addition of the amines, the resulting solution was diluted to afinal concentration of 0.2 M of the substituted ammonium salt.Osmolality was determined using a dew point osmometer. The properties ofresulting substituted alkylammonium sulfate salt solutions are shown inthe Table 1 below.

TABLE 1 Properties of various dialkylammonium and trialkylammoniumsulfate solutions Salt Osmolality, mmol/kg pH Dimethylammonium sulfate472 5.65 Dimethylethanolammonium sulfate 509 5.72 Diethylammoniumsulfate 519 5.85 Trimethylammonium sulfate 497 5.81 Triethylammoniumsulfate 559 5.33

Example 2. Preparation of Liposomes with Entrapped Dialkylammonium andTrialkylammonium Salts, and Loading of a Substance into these Liposomes

Distearoylphosphatidylcholine (DSPC), cholesterol (Chol), andN-(methoxy-poly(ethyleneglycol)-oxycarbonyl)-distearoylphosphatidylethanolamine (PEG-DSPE)(prepared from poly(ethylene glycol) with mol. weight 2,000) wereco-dissolved in chloroform in a molar ratio of 3:2:0.015, and thechloroform was removed at 55-60° C. by rotary evaporation. The driedlipid film was then hydrated in a solution of one of each dialkyl- ortrialkylammonium sulfates listed in Example 1 at 60° C. for 30 min. Thelipid suspension was extruded under pressure through two stackedpolycarbonate track-etched membrane filters with the pore size of 0.1 μm(Corning Nuclepore). The liposome size determined by quasielastic lightscattering method was approximately 110-120 nm. Unencapsulatedtrialkylammonium or dialkylammonium salts were removed from the externalmedium of the liposomes by gel filtration using a cross-linked dextrangel (Sephadex G-75, Amersham Pharmacia Biotechnology) column eluted withHEPES-buffered saline, pH 7.2-7.4, and the liposomes were collected in avoid-volume fraction of the column. Doxorubicin hydrochloride USP(lyophilized powder containing 5 weight parts of lactose per 1 part ofdoxorubicin) was added to the liposomes at a concentration of 150 μgdrug/μmol of liposome phospholipid. The mixture was incubated at 55° C.for 45 min, chilled on ice for 10 min, and unencapsulated drug wasremoved by gel filtration chromatography using a Sephadex G-75 columneluted with HEPES-buffered saline, pH 7.4. The presence of freedoxorubicin (characterized by the appearance of a slower moving redcolored band) was visually undetectable. The purified doxorubicin-loadedliposomes were analyzed for phospholipid and doxorubicin according toExamples 70 and 71 (spectrophotometric method), respectively. Theresulting drug loading efficiencies are shown in Table 2.

TABLE 2 Loading of doxorubicin in liposomes with entrapped solutions ofdialkyl- and trialkylammonium salts. Input drug/phospholipid ratio 150μg/μmol. Drug/phospholipid Entrapment ratio in liposomes EfficiencyLiposome-entrapped salt: (μg/μmol) (%) Trimethylammonium sulfate 140.74± 10.35 93.8 ± 5.7 Triethylammonium sulfate 163.81 ± 16.41 109.2 ± 11.6Diethylammonium sulfate 158.16 ± 18.34 105.4 ± 7.8 Dimethylethanolammonium sulfate 155.08 ± 8.51  103.4 ± 11.6

Example 3. Preparation of Liposomes Containing Various Dialkyl-,Trialkyl-, and Heterocyclic-Substituted Ammonium Sulfate Salts andLoading of Doxorubicin into these Liposomes

The substituted ammonium sulfate salt solutions were prepared as inExample 1 using commercially available alkyl-substituted,hydroxyalkyl-substituted and heterocyclic amines. Liposomes were formedas in Example 1, except that instead of the lipid film hydration step,the neat lipids were dissolved in ethanol (approximately 100 μl ofethanol for every 50 μmol of phospholipid) and mixed with thesubstituted ammonium salt solution at 60-65° C. so that the resultinglipid dispersion contained about 10 vol. % of ethanol.

Doxorubicin loading was accomplished by adding doxorubicin solution (2mg/ml in HEPES-buffered saline pH 6.5) to the liposomes at a ratio of155 μg drug/μmol liposome phospholipid (PL) and heating at 58° C. for 45min in a hot water bath. The resulting liposomes were separated from anyresidual unencapsulated doxorubicin and analyzed for drug and lipidcontent as in Example 1. The results are shown in Table 3.

TABLE 3 Loading doxorubicin into liposomes with entrapped stericallyhindered substituted alkyl, dialkyl-, trialkyl- and heterocyclicammonium salts solutions. drug load, Loading Amine used to prepareOsmolality, mg/mmol efficiency, substituted ammonium salt mmol/kgphospholipid % Trimethylamine 497 149.4 ± 7.9 96.4 ± 4.9 Triethylamine559 149.6 ± 6.9 96.5 ± 4.3 Dimethylethanolamine 509 163.1 ± 6.6 105.3 ±4.5  Dimethylamine 472 158.6 ± 7.4 102.3 ± 4.9  Diethylamine 519  156.7± 13.0 101.1 ± 8.5  Diisopropylamine 533 159.9 ± 6.2 103.2 ± 4.1 Tris(hydroxymethyl)- 423  179.9 ± 15.3 116.1 ± 11.5 minomethane1-Piperidineethanol 506 153.5 ± 7.1 99.0 ± 4.5 4-Methylmorpholine 465152.4 ± 9.8 98.3 ± 6.2 Piperidine 479  158.5 ± 12.5 102.3 ± 8.2 1-Methylpyrolidine 492  153.6 ± 12.3 99.1 ± 7.8 Dimethylpiperazine 378158.0 ± 6.5 101.9 ± 4.3 

Example 4. Preparation of triethylammonium polyphosphate (TEA-Pn)Solution

Linear sodium poly(phosphate) having 13-18 phosphate units per molecule(Phosphate glass; CALGON®, obtained from Sigma Chemical Company) wasdissolved in water to a concentration of about 1.3 M phosphate. Thesolution was passed through a column packed with 120 mL of sulfonatedpolystyrene-divinylbenzene copolymer cation exchange resin beads (Dowex50Wx8-200, Dow Chemical Co.) in the hydrogen form. The column waspre-equilibrated with aqueous 3-3.6 M HCl to bring the resin intohydrogen form, and washed with deionized water to neutral pH. Fifteen mlof the sodium polyphosphate solution was applied on the column andeluted with deionized H₂O. The column eluent was monitored using aconductivity detector. The column outflow corresponding to theconductivity peak was titrated with neat triethylamine to pH 5.5-6.0.The solution was analyzed for residual sodium by potentiometry using asodium-sensitive glass electrode and for phosphate content using aninorganic phosphate assay as in Example 1. The solution having residualsodium content of less than 1% was diluted to a final phosphateconcentration of 0.55 M. The solution typically has a TEA concentrationof 0.52-0.55 M, pH of 5.5-6.0, and osmolality of 430-480 mmol/kg

Example 5. Removal of Unentrapped Polyphosphate Salts from LiposomePreparations

Liposomes (120 nm in size) with entrapped fluorescent marker8-hydroxypyrene trisulfonate were prepared according to Kirpotin, etal., Biochemistry 36:66-75, 1997, and mixed with the solution of sodiumpolyphosphate. The mixture was loaded on size exclusion columnscontaining cross-linked dextran beads (Sephadex G-75), 6% agarose beads(Sepharose 6B-CL), or 4% agarose beads (Sepharose 4B-CL), all fromAmersham Pharmacia, and eluted with MES-Dextrose buffer (pH 5.5). Theeffluents were assayed for phosphate content using the phosphate assayof Bartlett (1959), and for the liposome content by spectrofluorometry.Of the studied gel-chromarography carries, Sepharose CL-6B providedcomplete separation of the polyphosphate from the liposomes at thesample/column bed volume ratio of 13.

Example 6. Preparing Solution of Triethylammonium Sucroseoctasulfate(TEA-SOS)

Sodium sucrose octasulfate (equivalent weight 144.8) is the sodium saltof sucrose derivative in which all hydroxyl groups have formed sulfuricacid esters. Sucrose octasulfate (SOS) sodium salt was purchased fromToronto Research Chemicals, Toronto, Canada, p/n S699020. Six grams ofsodium sucrose octasulfate was dissolved in 16.57 ml of deionized waterto give a final concentration of about 2.5 N of the sulfate groups. Thesolution was treated by ion exchange as in Example 4. The solution ofsucroseoctasulfuric acid obtained as an ion exchange column effluent wasthen titrated with neat triethylamine to pH 5.7 (neutralization point),and the pH and osmolality of the solution were determined. The resultingsolution had the calculated triethylammonium concentration of 0.643 M,pH 5.7, and the osmolality of 530 mmol/kg. The presence of residualsodium was undetectable by potentiometry (less than 0.1%).

Example 7. Liposomes Loaded with Irinotecan (CPT-11) Using SubstitutedAmmonium Salts: Preparation and In Vitro Drug Release in the Presence ofBlood Plasma

In this example, sulfate, citrate, pyrophosphate, triphosphate, andlinear polyphosphate (13-18 mer) were studied as anions in theliposome-entrapped substituted ammonium salt solutions. Phosphatepolymers were chosen because of their biodegradability and becausepolyphosphates are found naturally in the cells, as opposed to othersynthetic polymeric anions (polyacrylate, dextran sulfate, and thelike). Also, the viscosity of solutions of low molecular weightpolyphosphates was lower than that of other polymers, makingpolyphosphates more process-friendly.

The following materials were used for preparation of salt solutions.

1. Sodium polyphosphate, NaO—[PO₃Na]_(n)—Na, n=13-18, purchased fromSigma (Product No. P-8510, “Phosphate Glass, Practical Grade”, alsoknown as sodium hexametaphosphate or by the brand name CALGON);

2. Pentasodium tripolyphosphate, Na₅P₃O₁₀, purchased from Sigma (ProductNo. T-5883); 3. Tetrasodium pyrophosphate decahydrate, Na₄P₂O₇.10H₂O,purchased from Sigma (Product No. P-9146).

4. Ion exchange resins Dowex 50Wx4 (4% cross-linked sulfonatedpolystyrene resin, 100-200 mesh) purchased from Sigma (Product No.50X4-200) or Dowex HCR-W2 (8% cross-linked sulfonated polystyrene resin50-100 mesh) purchased from Sigma (Product No. I-8880) were usedinterchangeably. The resins were washed by decantation in the followingorder: three times with deionized water, twice with 1N HCl (3× excessover the resin by volume), three times with water, twice with 1N NaOH,three times with water, three times with 1N HCl, and three times withwater. After the decantation, the resins were in H⁺-form.

5. Trimethylamine (TMA), aqueous solution 40%, from Aldrich Chemical Co.(Product No. 43, 326-8). The concentration was established by acidtitration to be around 5.9 N.

6. Triethylamine (TEA), 99%, HPLC Grade, from Fisher (Product No.04884). The concentration by acid titration was 6.9-7.1 N.

Water was purified through reverse osmosis, ion exchange, and organicremoval to achieve organic free “16-18 MOhm” quality.

Aqueous solutions of pyrophosphate, triphosphate, and polyphosphatesalts were prepared by ion exchange method. Solutions of sodiumpolyphosphate (3 g in 25 mL of water), pyrophosphate (4 g in 27 mL ofwater), or polyphosphate (6.7 g in 30 mL of water) were loaded on thecolumn containing 100 mL (bed volume) of the ion exchange resin preparedas above. The column was eluted with water, and fractions werecollected. The fractions showing acidic pH (pH<3) were pooled.Triplicates of 0.5-mL aliquots of the pooled fraction containing thephosphate acid were diluted with 20 mL water and titrated with 0.100 NNaOH to the end point of pH 4.5-5.0 (Fisher analytical solution) todetermine normality. The pooled fractions after ion exchange weretitrated with trimethylamine (to obtain trimethylammonium salts) to pH5.4-5.5. After titration, the solutions were diluted, if necessary, toobtain a final concentration of trimethylammonium close to 0.5 N.

Trimethylammonium and triethylammonium sulfates were prepared bydiluting 1.39 mL of concentrated (17.9 M) sulfuric acid with 80 mLwater, and titrating the diluted solution with neat triethylamine oraqueous trimethylamine under the control of a pH-meter to equivalencepoint (pH 5.1-5.5). The volume was adjusted to 100 mL with water.

Trimethylammonium citrate solution was prepared by dissolving 1.572 g ofcitric acid monohydrate ACS from Sigma (Product No. C-1909) in 20 mL ofwater, and titrating the solution with aqueous trimethylamine to thepoint of equivalence. The volume was adjusted to 25 mL with water.

The solutions were filtered through a 0.2-μm cellulose acetate filterusing positive pressure. Osmolality and pH of the solutions was measuredusing a vapor pressure osmometer and glass-calomel electrode pH-meter,respectively. The normality of the anion in the phosphate solutions wasdetermined by blue phosphomolybdate spectrophotometric assay (seeExample 70) after acid hydrolysis (5 min. 100° C., 3N H₂SO₄). Anionnormality took into account only the acidic functional groups that aresubstantially ionized at pH 5.5. Cation normality was determined on thebasis of the added trialkylammonium base. The obtained solutions had thefollowing properties (Table 4):

TABLE 4 Properties of substituted ammonium salt solutions for CPT-11loading into liposomes. cation anion Osmolality Salt normality normalitypH (mmol/kg) TMA citrate 0.58 0.60 5.1 791 TMA sulfate 0.50 0.50 5.4 625TMA pyrophosphate 0.44 0.54 5.4 651 TMA triphosphate 0.57 0.68 5.4 695TMA polyphosphate 0.49 0.58 5.5 336 TEA sulfate 0.54 0.50 5.35 719

Cholesterol and DSPC were purchased from Avanti Polar Lipids, Alabaster,Ala., USA. PEG-DSPE (PEG mol. weight 2,000) was from ShearwaterPolymers, Huntsville, Ala., USA. DSPC, cholesterol, and PEG-DSPE in theweight ratio of 3:1:0.1 (molar ratio approximately 3:2:0.03) weredissolved in chloroform (Fisher; Optima grade, stabilized with amylene)at 60 mg/mL of DSPC. The solution was dispensed into Pyrex tubes at 30mg of DSPC (0.5 mL) per tube and slowly evaporated at reduced pressureusing rotary evaporator at 60° C. The lipid films were dried undervacuum (100 micron mercury, oil pump) for 30-60 minutes at roomtemperature.

Dry lipid films were hydrated by gentle shaking in the above aqueoussalt solutions at 60° C. for 15-20 minutes. The lipids formed a milkysuspension (multilamellar vesicles). This milky suspension was subjectedto five cycles of freezing in the mixture of dry ice and isopropanol(−80° C., 3 minutes) and thawing in a water bath at 60° C. for 3minutes. Then, the lipid suspension was extruded 10 times(double-strokes) through two stacked polycarbonate membrane filters(Nucleopore, Whatman, pore size 0.1 μm) using a manually operatedreciprocating extruder (Avanti Polar Lipids) heated at 60° C.

The extruded liposomes were kept at 60° C. for five minutes and quenchedin ice water (0-4° C.) for five minutes. Then, the liposomes wereseparated from the gradient-forming salt solution into the loadingbuffer IVIES-Dextrose (50 g/L of Dextrose ACS, 0.975 g/L of2-(N-morpholino)-ethanesulfonic acid (IVIES), and sufficient amount of5M NaOH to bring the pH to 6.4) by gel-chromatography on Sephadex G-75.Liposomes appear in the void volume fraction (approximately 30% of thecolumn bed volume).

CPT-11 (Irinotecan hydrochloride) preparation containing 0.860 mg of theCPT-11 base per 1 mg of the solid was dissolved in 0.001N HCl to make astock solution of 16.5 mg/mL CPT-11 base. This solution was mixed withthe liposomes in IVIES-Dextrose buffer to achieve the ratio of 150 μgCPT-11 per 1 μmol of liposome phospholipids. The mixture was incubatedat 55° C. in a water bath, with occasional gentle shaking (approximatelyevery five minutes) for 30 minutes, then quickly chilled in ice water(0-4° C.). The liposomes were separated from the unencapsulated drug bygel-chromatography on Sephadex G-75, using IVIES-Dextrose as eluent. Theencapsulated drug was determined by a spectrophotometric assay (Example71), and the phospholipids determined using an extraction assay (Example70).

In vitro drug release from so obtained CPT-11-loaded liposomes in thepresence of 50% human plasma was studied as follows. Frozen human donorplasma was thawed as 37° C., centrifuged at 14,500 g for 10 minutes, andfiltered through a 0.45-μm cellulose acetate syringe filter. Theliposome preparations with loaded CPT-11 were sterilized by passagethrough 0.2-μm surfactant-free cellulose acetate (SFCA) sterile syringefilter. 0.5-mL of the liposomes were mixed with 0.5 mL of plasma insterile 1.5-mL copolymer Eppendorf tubes, sealed, and incubated on arocking platform at 37° C. for 24 hours. Blank sample contained 0.5 mLof sterile IVIES-Dextrose instead of liposomes. The liposomes wereisolated by gel-chromatography on a beaded cross-linked 2% agarose gel(Sepharose CL-2B, Pharmacia; 10 mL bed volume) using 144 mM NaCl, 5 mMHEPES-Na, pH 7.4 buffer (HBS-5). The liposomes appeared at the voidvolume fraction, while plasma proteins and released drug (if any) wereretarded by the gel. The liposome fractions were assayed for CPT-11 andphospholipids, and the drug/phospholipids ratio (output ratio) wasdetermined. Readings of the blank samples (plasma only) were subtractedfrom the readings of the liposome-containing samples. Percent of thedrug remaining in the liposomes after the incubation was determined bydividing output drug/lipid ratio by the input drug/lipid ratio(drug/lipid ratio prior to incubation with plasma). The loading andrelease data are summarized in Table 5.

TABLE 5 Loading of CPT-11 into liposomes with tertiary alkylammoniumsalts and in vitro release of the drug in the presence of human plasma.Before incubation with plasma After incubation with plasma Entrappedsalt drug/lipid encapsulation drug/lipid drug remaining solution ratioefficiency (%) ratio encapsulated (%) TMA sulfate 127.2 ± 5.6 84.8 ± 3.8132.1 ± 6.9 103.8 ± 10.0 TMA pyrophosphate 136.2 ± 9.0 90.8 ± 6.0 132.3± 5.0  97.1 ± 10.1 TMA triphosphate 132.9 88.6 129.2 97.3 TMA-Pn 134.4 ±9.3 89.6 ± 6.2 135.0 ± 7.4 100.4 ± 12.4 TEA sulfate 131.1 ± 6.5 87.4 ±4.4 125.2 ± 5.0 95.5 ± 8.6

Example 8. In Vivo Stability of the Liposomes Loaded with CPT-11 UsingPyrophosphate, Triphosphate, Polyphosphate, Citrate, and SulfateTrialkylammonium Salts

While camptothecin liposomes may show acceptable drug leakage in bloodplasma in vitro, the drug may leak more quickly in the blood circulationin vivo. Therefore, a panel of CPT-11 liposome formulations was screenedfor drug stability in the blood circulation in vivo using a single timepoint assay in mice.

The liposomes were prepared and loaded with CPT-11 as described inExample 6, with the following modifications. Instead of using PEG-DSPEfrom Shearwater Polymers, we used similar PEG-DSPE from Avanti PolarLipids. To afford quantification of the liposome lipid matrix in theblood/tissue samples, a non-exchangeable radioactive label,[³H]-Cholesteryl hexadecyl ether ([³H]-CHE; (Amersham, USA) was added tothe chloroform solution of the lipids in the amount of 0.25 mCi/mmol ofphospholipids. The lipid solutions were dispensed into Pyrex tubes at 12mg of DSPC/tube, and lipid films were formed by rotaryevaporation/vacuum drying. Lipid films were hydrated in 0.7 mL of thegradient-forming substituted ammonium salt solutions. Lipidconcentration in the liposomes with entrapped phosphate-containing saltswas determined by radioactivity scintillation counting. The preparationswithout entrapped phosphate-containing salts were also assayed forphospholipids without extraction as described in Example 70, and used aslipid radioactivity standards. Portions of the liposome-drug mixturesprepared for the loading were saved and assayed to confirm thepre-loading ratio of the added CPT-11 to the liposome lipid prior toloading (“input ratio”). Volume-averaged mean and standard deviation ofthe liposome size distribution were determined by quasi-elastic lightscattering (QELS) using Gaussian model. The properties of theseliposomes are summarized in Table 6.

TABLE 6 Characterization of CPT-11 loading into [³H]-CHE-labeledliposomes for in vivo stability study Entrapped salt drug/lipid ratiodrug/lipid ratio loading liposome size, solution before loading afterloading efficiency (%) (mean ± SD) nm TMA citrate 159.2 ± 3.5 156.7 ±3.6 98.5 ± 4.4 122.1 ± 25.3 TMA sulfate 156.1 ± 2.5 156.1 ± 3.1 100.0 ±3.6  122.2 ± 28.4 TMA pyrophosphate 164.6 ± 5.8 156.6 ± 4.3 95.2 ± 6.0121.1 ± 19.9 TMA triphosphate 163.6 ± 5.7 156.0 ± 3.2 95.3 ± 5.3 122.4 ±12.9 TMA polyphosphate 170.5 ± 8.0 162.4 ± 4.0 95.3 ± 6.8 123.0 ± 12.7TEA sulfate  153. ± 3.3 154.9 ± 4.9 101.0 ± 5.3  121.1 ± 18.0

Six-week-old female CD-1 mice (Charles River) received tail veininjections of these liposomal CPT-11 formulations at the dose of 10mg/kg (0.2 mg CPT-11/mouse) in duplicate. Eight hours later, the micewere anesthetized and exsanguinated through open heart puncture. Theblood was collected into heparinized syringes (10-20 μl of 1000 U/mlheparin USP) and transferred into weighed tubes containing 0.4 ml of thephosphate-buffered physiological saline solution (PBS) containing 0.04%EDTA (Gibco BRL), kept on ice. The tubes were weighed to determineweights of the blood samples, blood cells were separated bycentrifugation at 9,000 g for 5 minutes, and supernatants containingPBS-diluted plasma, were saved for drug and liposome lipid assays.CPT-11 was quantified by fluorometric assay (Example 71). Liposome lipidwas quantified by quenching-corrected radioactivity scintillationcounting. The liposome and phospholipid-radioactivity standards werecounted in parallel with the plasma samples. Percent of the drug thatremained encapsulated was calculated by dividing the drug/radioactivityratio in the plasma samples by the drug/radioactivity ratio of theinjected liposomes. Due to the fast elimination of free CPT-11 from theblood (see Example 69) and the known stability of the [³H]-CHE againstlipid exchange, the assays' readings were considered indicative of theblood content of liposomal CPT-11 and lipid. Percent of injected lipiddose (% I.D.) remaining in the circulation was calculated assuming 100%of the injected bolus entered the circulation; blood volume being 6.3%of the mouse body weight, and hematocrit of 45%. The results aresummarized in Table 7.

TABLE 7 In vivo stability of CPT-11-encapsulation and circulationlongevity of CPT-11-loaded liposomes in mice at a single point (8 hours)post injection. % I.D., % of injected dose. Liposome- Drug/lipid ratio,% Liposome lipid, % entrapped salt of pre-injection value I.D. in theblood TMA citrate 80.2 ± 7.8 18.8 ± 3.4 TMA sulfate 70.1 ± 4.8 23.6 ±1.8 TMA pyrophosphate 67.3 ± 9.2 23.2 ± 3.1 TMA triphosphate 70.6 ± 6.024.9 ± 8.2 TMA polyphosphate 107.5 ± 8.9  24.3 ± 3.4 TEA sulfate  76.6 ±13.1 23.6 ± 0.1

All preparations showed the level of drug encapsulation after 8 hours inthe blood in vivo at 70-80% of the pre-injection level, while theliposomes containing polyphosphate were the most stable (drugencapsulation remains at about 100%).

Example 9. Blood Pharmacokinetics of CPT-11 Liposomes Prepared UsingTriethylammonium Polyphosphate

The formulation of liposomal CPT-11 using triethylammonium polyphosphatesalt was prepared as outlined in Example 3. The lipids—DSPC,cholesterol, and N-(methoxy-poly(ethylene glycol) (M.w.2000)-oxycarbonyl)-DSPE (PEG-DSPE) (all from Avanti Polar Lipids,Inc.)—were combined as dry powders in the molar ratio of 3:2:0.015 anddissolved in 100% ethanol (USP grade, approx. 0.15 mL/100 mg of thelipids) at 62-65° C. For pharmacokinetic studies, ³H-cholesterylhexadecyl ether (³H-CHE, obtained from Amersham Pharmacia) was added tothe lipids in the amount of 0.5 mCi/mmol of phospholipids as anon-exchangeable radioactive lipid label. The aqueous solution of TEA-Pn(0.5 M triethylammonium, pH 5.7-6.2) was prepared as in Example 4.TEA-Pn solution (10 times the volume of added ethanol) was mixed withthe lipid solution at 60-65° C. and stirred at this temperature until ahomogeneous milky suspension of multilamellar vesicles was formed. Thissuspension was extruded 15 times through 2 stacked polycarbonatetrack-etched filters (Corning Nuclepore) with the pore size of 100 nmusing argon pressure extruder (Lipex Biomembranes) at 60-65° C., andresulting unilamellar liposomes were quickly chilled in ice and then letattain ambient temperature. Ethanol and unincorporated polyphosphatesalt were removed by gel chromatography on Sepharose CL-4B column elutedwith MES-Dextrose buffer (5 mM IVIES, 50 g/L dextrose, pH adjusted to6.5 with NaOH).

A stock solution of CPT-11 (Irinotecan hydrochloride) containing 20mg/mL Irinotecan base in water was added to the liposomes at adrug/lipid ratio of 150-200 mg/mmol phospholipids, and the mixture wasincubated with occasional agitation for 45-60 minutes at 60-62° C. Theincubation mixture was quickly cooled down and incubated for 10 minutesat 0° C., then allowed to attain ambient temperature. 1/20 of the volumeof 2.88 M NaCl was added to adjust to physiological ionic strength andimprove the removal of membrane-bound CPT-11 (as opposed to the drugencapsulated within the liposome interior). Unencapsulated drug wasremoved by gel chromatography on Sephadex G-25 or G-75 column (AmershamPharmacia) eluted with HBS-6.5 buffer (5 mM2-(4-(2-hydroxyethyl)-piperazino)-ethylsulfonic acid (HEPES), 144 mMNaCl, pH 6.5). Liposome fractions eluted in the void volume werecombined, sterilized by 0.2 micron filtration, and stored at 4-6° C.before use. The liposomes were characterized by lipid concentration,drug concentration, and particle size as in Example 7. The liposomes hadthe average size of 108 nm and CPT-11 content of 139±18 mg of CPT-11base per mmol of phospholipids.

The longevity of the liposome lipid and liposome drug in the blood andthe characteristics of drug release from the liposomes in vivo werestudied in female Sprague-Dawley rats (190-210 g) with indwellingcentral venous catheters. The rats were injected with a 0.2-0.3 mL bolusof ³H-CHE-labeled Irinotecan liposomes (0.05 mmol phospholipids, or 7-8mg CPT-11 per kg of the body weight). Blood samples (0.2-0.3 mL) weredrawn at various times post injection using heparin-treated syringe. Thewithdrawn blood volume was replenished using phosphate bufferedphysiological saline. The blood samples were diluted with 0.3 ml ofice-cold PBS containing 0.04% EDTA, weighed, and the blood cells wereseparated by centrifugation. The supernatant fluids were collected andassayed for CPT-11 using the fluorometric procedure of Example 71, andfor the liposome lipid label by scintillation radioactivity countingusing conventional methods. The liposome preparations with known drugand ³H-CHE-lipid concentration were used as standards. Radioactivitystandards contain equal amount of diluted rat plasma to account forquenching. The amount of CPT-11 and the liposome lipid in the blood wascalculated assuming the blood volume in ml as 6.5% of the body weight ingrams, and the hematocrit of 40%. The total amount of the lipid and thedrug in the blood was expressed as % of injected dose (% I.D., % ID) andplotted against post-injection time. The percent of drug remaining inthe liposomes was calculated by dividing the drug/lipid ratio in theblood samples by the drug/lipid ratio of the injected liposomes (takenas 100%). Because the plots generally showed good agreement withmonoexponential kinetics (linearity in semi-logarithmic scale), bloodhalf-lives of the drug, the lipid, and of the drug release from theliposomes, were calculated from the best fit of the data tomonoexponential decay equation using the TREND option of the MicrosoftEXCEL computer program (Microsoft Corp., USA). The results are shown onFIG. 1. From the best fit parameters, the blood half-lives for lipid anddrug were 16.4 hours and 6.61 hours, respectively. Under theseconditions, free CPT-11 clears from the circulation very rapidly (seeExample 69).

The blood drug/lipid ratio revealed biphasic character of the CPT-11release from the liposomes (FIG. 2). In the first 24 hours, the releaseof drug followed was linear over time (R=0.992) giving evidence forzero-order release kinetics. Only after about 75% of the drug wasreleased at 24 hour time point, further release became non-linear. For24 hours, the liposomes released the drug at a constant rate of about3.6% of the initial load/hour. Thus, 50% of the drug was released overthe period of approximately 14 hours. Zero-order release of the drug isan attractive quality in sustained release formulations, as the rate ofdrug release remains constant over time.

Example 10. Antitumor Efficacy of CPT-11 Liposomes Prepared UsingTriethylammonium Polyphosphate Against Breast Cancer Xenografts in NudeMice

Antitumor efficacy of CPT-11 liposomes was studied in the model of humanbreast carcinoma BT-474, an estrogen-dependent ductal adenocarcinomathat over-expresses C-ErbB2 (HER2) receptor. BT-474 cells were obtainedfrom American Type Culture Collection (Rockville, Md.). A BT-474sub-line with higher tumor growth rate was established from afast-growing xenograft tumor nodule raised as described below. The cellswere propagated in vitro in RPMI-1460 medium with 10% fetal calf serum,0.1 mg/mL streptomycin sulfate, and 100 U/ml Penicillin Gin T-150flasks, and split 1:3 every week. NCR nu/nu female mice (4-6 week old;Taconic Farms) were subcutaneously implanted (at the base of tail) with60-day sustained-release 0.72-mg 1713-estradiol pellets (InnovativeResearch of America, Inc.), and in two days were inoculatedsubcutaneously in the upper back area with 0.1 mL suspension containing2×10⁷ BT-474 cells in cell growth medium. The tumor progression wasmonitored by palpation and caliper measurements of the tumors along thelargest (length) and smallest (width) axis twice a week. The tumor sizeswere determined twice weekly from the caliper measurements using theformula (Geran, R. I., et al., 1972 Cancer Chemother. Rep. 3:1-88):

Tumor volume=[(length)×(width)]/2

At day 13 after inoculation, the tumor reached an average size of 200mm³ and the animals were randomly assigned to three groups of 13-15animals.

Liposomal CPT-11 was prepared as in Example 8 (drug/phospholipid ratio192 mg/mmol; average liposome size 86.8 nm). Free and liposomal CPT-11were diluted with IVIES-dextrose vehicle to 5 mg/ml of CPT-11 base Theanimals were injected via the tail vein with free CPT-11, liposomalCPT-11, or vehicle only on days 14, 18, 21, and 25 post tumorinoculation. The drug-containing formulations were given at the dose of50 mg CPT-11/kg per injection, which is the average of the dosesreported in the literature for the CPT-11 studies in rodent tumormodels.

To assess treatment-related toxicity, the animals were also weightedtwice weekly. The observations were made until day 60 post inoculation,at which time the estrogen supplementation pellet was exhausted. Averagetumor volumes across the groups were plotted together and compared overtime. As shown in FIG. 3, while free CPT-11 reduced the rate of tumorgrowth, in the group that received liposomal treatment the tumorsregressed dramatically. While at day 36 in the control group the tumorsreached the maximum allowable size averaging 3,500 mm³, and at day 46 inthe group treated with free drug the tumors were about 1,000 mm³ ataverage, at the same time point none of the animals in theliposome-treated group had a palpable tumor.

The treatment related toxicity was assessed by the dynamics of animals'body weight (FIG. 4). Neither group revealed any significant toxicity.The weight of the animals in the control group was consistentlyincreasing. There was a slight decrease in the average body weight ofthe animals receiving liposomal CPT-11, by about 3.3%, on the day of thelast treatment. This weight loss was reversed, however, and the animalsreached their expected weight. This decrease in the mean body weight wasnot statistically significant by Student's t-test compared topretreatment weight (p=0.274). Thus, all treatments were toleratedwithout significant toxicity.

Thus, the liposome formulation of CPT-11 obtained by loading of the drugvia pre-entrapped sterically hindered substituted ammonium salt(triethylammonium) of a polyanionic, biodegradable polymer(polyphosphate) showed extended blood life, sustained releasecharacteristics, and increased antitumor activity in the studied tumormodel without an appreciable increase in toxicity.

Example 11. Comparative Assessment of CPT-11 Loaded Liposomes PreparedUsing Pre-Entrapped Triethylammonium Salts: Effect of Liposome Size,Drug/Lipid Ratio, and the Nature of Pre-Entrapped Anion

Two prototype formulations of CPT-11-loaded liposomes were prepared, oneusing the liposomes with pre-entrapped TEA-Pn, and the other withpre-entrapped TEA-SOS. Preparation of these liposomes included thefollowing manufacturing steps.

1) Combining the lipids by co-dissolving in ethanol. The lipid matrixcomposition consisted of 1,2-Distearoyl-SN-phosphatidylcholine (DSPC)(Mol. wt. 790) 3 molar parts (59.8 mol. %); Cholesterol (Chol) (Mol.weight 387) 2 molar parts (39.9 mol. %); andN-(omega-methoxy-poly(ethyleneglycol)-oxycarbonyl)-1,2-distearoylphosphatidyl ethanolamine (Mol.weight 2787) (PEG-DSPE) 0.015 molar parts (approx. 0.3 mol. %). DSPC andPEG-DSPE were purchased from Avanti Polar Lipids, Birmingham, Ala.Cholesterol (highest purity grade) was purchased from Calbiochem. Drylipids were weighed with the accuracy of ±0.1 mg in a borosilicate glasscontainer and combined with absolute ethanol at the ratio suitable forthe lipid dispersion step below. Because of the high transitiontemperature of DSPC (55° C.) the dissolution was typically performed at55-60° C. until clear solution was obtained.

2) Preparing the TEA-Pn and TEA-SOS solutions. Sodium polyphosphate(n=13-18) was from Sigma Chemical Co., p/n P 8510. Sodium sucroseoctasulfate was purchased from Toronto Research Chemicals, Toronto,Canada, p/n S699020. The salts were weighed down and dissolved in waterto provide 1.2-2.5 N solutions. Anion exchangers Dowex 50Wx8-200 orDowex HCR-W2 in H⁺-form (available from Sigma) were used to convert thesodium salts into free acids. Before the first use, the resins werewashed by stirring with 3 volumes of the following solutions, followedby decanting: (1) 1.0-1.2 M aqueous HCl 2 times; (2) Water 2 times; (3)1.0-1.2 M aqueous NaOH 2 times; (4) Water 2 times; (5) 1.0-1.2 M aqueousHCL, 2 times. The suspension of washed resin in water was packed undergravity flow in a suitable size chromatography column to have at least 8mL of the packed resin for each mL of the sodium salt solutions. Theresin was further equilibrated by passage of 2 column volumes of 3.0-3.6M aqueous HCL, followed by 5 column volumes of water or until theconductivity of the eluate falls below 1 micro-S. After use, the columnswere regenerated by sequential passage of: 1.0-1.2 M HCl—3 columnvolumes; 3.0-3.6 M HCl—2 column volumes; water—at least 5 columnvolumes, or until the conductivity of the eluate falls below 1 μS, andstored under 0.2-um filtered water at room temperature. The Pn and SOSsodium salt solutions were applied on the drained surface of the column(approximately 1 ml for each 4 ml of the packed resin volume) andallowed to flow under gravity at the rate of about 1-2 ml/min for theresin bed size of 75-150 mL. The column was eluted with water. Theeluate was tested for conductivity. The fractions with 10 mS or higherconductivity were collected. If more concentrated solutions of polyacidsare required, the collection can start at 20-50 mS, but at the expenseof somewhat higher loss of the gradient-forming salt. In the case ofpolyphosphoric acid, the collected solution is kept refrigerated (0-4°C.) until the amine titration step because of the hydrolytic instabilityof the phosphodiester bond at low pH. The collected eluates would have apH of less than 0.7 (typically about 0.4) and conductivity of about120-200 mS. Optionally, the amine titration step is performed withoutdelay because the stability of polyphosphate at low pH. HPLC-gradetriethylamine (99.5+% purity) from Fisher, p/n 04884 was used to titratethe acid solutions obtained from ion exchange. The normality of neat TEAwas determined by potentiometric titration. 0.100-mL Aliquots of TEA(0.100 ml) were taken into 20 ml of water in triplicate. The aliquotsare titrated with 0.1 N HCl standard solution to the pH end point (glasselectrode) of 5.5-6.0. The calculated normality (7.07 N) was close tothe theoretical value of 7.17N. A measured volume of the polyphosphoric(Pn) acid or sucrose octasulfuric (SOS) acid solution was titrated withneat TEA under the control of pH (glass) electrode. Thorough stirringwas required to disperse the amine. Titration endpoint was pH 5.6-6.2.The volume of added TEA was accurately recorded. The volume of titratedsolution was measured, and the concentration of TEA was calculated onthe basis of the added TEA volume and normality. Water was added asnecessary to adjust the TEA concentration to required 0.55±0.05 N or0.65±0.03 N, as indicated below. The amount of residual sodium in theobtained TEA-Pn or TEA-SOS solutions was determined by potentiometryusing sodium-selective glass electrode (Corning). One mL of the solutionwas diluted with 19 mL of water, and the sodium concentration wasdetermined using the increment method according to the electrodemanufacturer's manual. The amount of residual sodium was less than 1 mM,typically less than 0.5 mM. The obtained TEA-Pn or TEA-SOS solutionswere passed through 0.2 μm cellulose acetate sterile filter usingpositive pressure feed. The final pH and osmolality of the solutions wasmeasured and recorded. We use pH calomel micro-combination all-glasselectrode for pH measurements, and vapor pressure/dew point osmometerfor osmolality measurements. The solutions were stored refrigerateduntil use.

3). Preparing lipid dispersion in the gradient-forming buffer by mixingof ethanolic solution of the lipids with the gradient-forming buffer.The lipids were dispersed in the gradient-forming salt solution usingethanol mixing method. All steps were performed at 60-65° C. The lipidswere dissolved in 100% Ethanol USP at a concentration of about 0.5-0.6 Mof DSPC in a chemical resistant glass pear-shaped flask or tube. Thegradient-forming salt solution (TEA-Pn or TEA-SOS) was pre-warmed to60-65° C. and added to the ethanolic lipid solution at once, and thecomponents were thoroughly mixed by swirling and/or vortexing. The finalamount of ethanol was about 10 vol. %. For preparations of the scale inexcess of 0.1 mmol phospholipid, the resulting suspension was placed ona rotary evaporator at 60-65° C. and vacuumized with rotation until theevolution of ethanol stopped, as manifested by the end of foamformation. For the scale of 0.1 mmol phospholipid or less, ethanol wasnot removed from the lipid dispersion at this step. The resulting lipidsuspensions were kept at 60-65° C. and used promptly for the extrusionstep.

4). Sequential extrusion of the lipid dispersion through defined poremembranes. For the lipid suspension volumes up to 1 mL we used amanually operated reciprocating extruder supplied by Avanti PolarLipids. The extruder is charged with 19 mm track-etched filter membranesand thermostatted by virtue of a metal heating block. For the volumesfrom 1 to 10 mL, we used a thermostatted, gas pressure operated,unidirectional flow extruder from Lipex Biomembranes. The extruder ischarged with 25 mm filter membranes. The lipid suspensions wererepeatedly extruded at 60-65° C. using manual feed or argon gaspressure, as appropriate, through a series of 2 stacked polycarbonatemembrane filters (the filters from Corning-Nuclepore and Osmonics Corp.were equally suitable) having nominal pore sizes of 100 nm, 80 nm, or 50nm. Where the effect of liposome size was of interest, the extrusion wasstopped at 100 nm, 80 nm, or 50 nm step. The exact type of filters usedand number of extrusions is indicated below for each experiment. Theextruded liposomes were kept at 60-65° C. for about 15 min. and quicklycooled down to 2-4° C. in an ice bath. After about 15 min. in the icebath, the liposomes were allowed to reach room temperature.

5). Removal of extraliposomal gradient-forming buffer and transfer ofthe liposomes into a drug-loading buffer. Non-encapsulatedgradient-forming salt was removed, and the liposomes were transferredinto the drug loading buffer using size exclusion chromatography (SEC).Tangential flow filtration, hollow fiber dialysis, of other scalablestep can be used in scale-up manufacture. It is advantageous to ensurethe complete removal of the extraliposomal polyanion by treatment of theliposomes with an anion-exchange resin (e.g., Dowex-1 or Dowex-2quaternary ammonium cross-linked polystyrene beads). Drug-loading buffercontained 50 g/L anhydrous Dextrose USP, and 5 mM tissue-culturecertified HEPES in water, adjusted to pH 6.5 with NaOH. The buffer wasvacuum-filtered through 0.2 micron Nylon filter (Whatman). The extrudedliposomes were chromatographed on a column with Sepharose CL-4B(Pharmacia) and eluted with the drug-loading buffer. The liposomesappeared in the void volume fraction and were collected, based on theeluate turbidity, in the volume of about 2× of that applied. The elutedliposomes were assayed for phospholipid concentration according toExample 70, particle size by QELS, and stored at 4-6° C.

6) Incubation of liposomes with the drug. Stock solution of CPT-11(Irinotecan Hydrochloride) was prepared immediately before mixing withthe liposomes by dissolving Irinotecan Hydrochloride in water to achieveconcentration of 20 mg/mL drug base. The pH of the solution was between4.0 and 5.0. The drug solution was filtered through 0.2 micronpolyethersulfone (PES) sterile filter using positive pressure. Aliquotsof the liposomes in the drug loading buffer produced at the step 5 abovewere mixed at room temperature with the stock Irinotecan solution toachieve the drug/lipid input ratio in the range of 0.15-0.55 g of drugfor mmol of liposome phospholipid. Particular input drug/lipid ratiosare indicated below, where appropriate. The pH of the mixtures wasadjusted to 6.5 with 1 M NaOH, the mixtures in glass vials wereincubated on the thermostatted water bath at 58-62° C. with slowagitation for 30-45 min, quickly cooled down in ice-water bath (0-2°C.), and left at this temperature for 15 min. Then the liposomes wereallowed to warm up to room temperature for the next step (removal ofunencapsulated drug and transfer into the storage buffer). This stepresulted in the encapsulation efficiency of more than 95%, typically98-100% in the whole range of studied drug/lipid ratios.

7). Removal of unencapsulated CPT-11, transfer of the liposomes into thestorage buffer, final filtration, and storage. Unencapsulated drug wasremoved and the liposomes were transferred into the storage buffer usingsize exclusion chromatography. The storage buffer contained 20 mM HEPES,135 mM NaCl, pH 6.5 (adjusted with NaOH) in water, and was 0.2-micronvacuum-filtered before use. Gel-chromatography on Sephadex G-75(Amersham Pharmacia Biotech) was performed essentially as describedunder Step 2 above. CPT-11 liposomes eluted from the column (void volumefraction) were assayed for liposome phospholipid and CPT-11 (byspectrophotometry, see Examples 70 and 71), and volume-weighted meanparticle size by QELS. The drug concentration was adjusted, ifnecessary, to be in the range of 2.0-4.0 mg/mL. The liposomes werefiltered through 0.2 micron polyethersulfone sterile filters andaseptically dispensed into sterile polypropylene vials (CorningCryo-Vials) or PTFE-lined screw-cap borosilicate 4-mL glass vials toapproximately 70-80% of the vial volume. The vials were asepticallyclosed (in air), labeled, and stored at 4-6° C.

Example 12. Effect of Drug/Lipid Ratio on the Drug Loading Efficiencyand In Vivo Drug Retention of TEA-Pn-Containing Liposomes

Liposomes with entrapped aqueous 0.65N solution of TEA-Pn, pH 6.1,osmolality 531 mmol/kg, were prepared following the procedure of Example11. The lipid dispersion was extruded ten times through two stacked 100nm pore size polycarbonate filters. Liposome lipid matrix also included[³H]-CHE at 0.5 mCi/mmol phospholipid. The liposome size before drugloading was 98.5+34.3 nm. The liposomes were loaded at initialdrug-to-phospholipid ratios of 200, 300, 400, and 500 mg CPT-11/mmolphospholipid. The drug and phospholipid amounts in the liposomes weredetermined by spectrophotometry according to Example 71, and byphospholipid extraction-digestion-blue phosphomolybdate assay of Example72, respectively.

To evaluate in vivo drug release rate, the method of Example 8 wasfollowed. The liposomes were injected via tail vein into 6-week-oldfemale Swiss Webster mice (body weight 18-22 g) at a dose of 5 mgCPT-11/kg. At 8 and 24 hours post injection, the mice, in groups of 3,were anesthetized, and exsanguinated via open heart puncture. The bloodwas mixed with 0.4 mL of ice-cold 0.04% EDTA in PBS, the blood cellswere separated by centrifugation, and the plasma concentration of CPT-11was measured by spectrofluorometry as described in Example 71. Lipid wasdetermined by measuring the amount of [³H]-CHE using quenching-correctedliquid scintillation counting, and the amount of drug retained in theliposomes was calculated by dividing the determined drug/lipid ratio bythe drug/lipid ratio in the injected liposomes. Because of the fastblood clearance of free CPT-11, resulting in low blood level, we assumedthat all assayed drug was in the liposomal form.

The results are presented in Table 8. The differences between drugretention among the groups were not statistically significant. As aresult of these studies, we concluded that increasing the drug load upto 500 mg/mmol will not adversely affect drug loading or in vivostability. This ratio was adopted for further studies.

TABLE 8 The effect of drug/lipid ratio on the drug loading and in vivodrug retention in Irinotecan TEA-Pn liposomes (average ± standarddeviation). Drug/lipid ratio, Drug remaining in the liposomes, mg/mmolphospholipid % of pre-injection value Input Output % loaded After 8hours After 24 hours 200 208.4 104.2 54.6 ± 9.9   9.72 ± 2.23 300 286.395.4 85.2 ± 14.3 14.52 ± 2.51 400 348.8 87.2 81.5 ± 18.3 17.31 ± 6.14500 518.9 103.8 66.8 ± 19.6 13.47 ± 1.44

Example 13. Drug Loading Efficiency of CPT-11 Loading intoTEA-SOS-Containing Liposomes: Effect of Liposome Size and In Vivo DrugRetention in Mice

Liposomes with entrapped solutions containing prepared as in Example 11using gradient forming solution having 0.643 N TEA-SOS, pH 5.7,osmolality 530 mmol/kg. Lipid dispersion was extruded ten times throughtwo stacked polycarbonate filters with the pore size of 50 nm, 80 nm, or100 nm. Liposome lipid matrix also included [³H]-CHE at 1.5 mCi/mmol ofliposome phospholipid. The liposome size was determined by dynamic lightscattering. The liposomes were loaded with CPT-11 at initialdrug-to-phospholipid ratios of approximately 550 mg Irinotecan/mmol ofphospholipid. The drug loaded liposomes were sized by QELS and assayedas described in Examples 70 and 71.

Female Swiss Webster mice (8-10 weeks, average 27-30 grams) wereinjected via tail vein with these CPT-11 liposome formulations at a drugdose of 10 mg/kg. The mice were sacrificed at 24 h and the blood wascollected and assayed for CPT-11 and liposome lipids as in Example 11.The results are summarized in Table 9.

TABLE 9 Irinotecan loading and in vivo drug retention in TEA-SOSliposomes. Extrusion Drug remaining in the membrane Liposome Drug load,mg liposomes after 24 pore size, size, nm Irinotecan/mmol hours in mice,% of nm mean SD phospholipid pre-injection value 50 87.6 ± 28.1 579.3 ±24.2 79.2 ± 3.8 80 98.5 ± 15.1 571.1 ± 69.7 82.6 ± 2.1 100 110.8 ± 25.2 567.7 ± 37.7 86.2 ± 2.7

Surprisingly, the liposomes with triethylammonium salt of sucroseoctasulfate, a non-polymeric polyanionized organic hydroxylated organiccompound (sugar), provided dramatically better (4-5 fold) in vivo drugretention in liposomes compared with similar liposomes with apolyanionic polymer (polyphosphate).

Example 14. Blood Pharmacokinetics of CPT-11-Loaded SOS-TEA Liposomes inRats

Liposomes (100 nm extrusion membrane pore size) were prepared asdescribed in Example 12 The liposomes was administered intravenously ata dose of 10 mg CPT11/kg to two nine-week-old female Sprague Dawley rats(Harlan) (body weight about 200 g) with indwelling central venouscatheter at a dose of 10 mg CPT-11/kg (17.6 μmol of phospholipids/kg).Blood samples were taken at prescribed time points and analyzed for drugand liposome lipid content as in Example 9. The data were expressed asthe % injected lipid dose/ml of plasma and the % drug retained insidethe liposome at each time point, plotted against post injection time,and half-lives for liposome lipid, as well as half-lives for drugrelease from the liposomes, were calculated by best fit to amonoexponential kinetic model (FIG. 5). The half-life of drug releasefrom CPT-11 loaded TEA-SOS liposomes was 56.8 hours, much longer thanthat of the similar TEA-Pn liposomes.

Example 15. Antitumor Activity of Free CPT-11, and CPT-11 Encapsulatedinto TEA-Pn and TEA-SOS-Containing Liposomes in Athymic Nude MiceBearing Subcutaneous Xenografts of Human Colon Carcinoma (HT-29)

The liposomes were prepared as in Example 11 using TEA-Pn solution with0.65 M TEA, pH 6.1, and osmolality 531 mmol/kg, or TEA-SOS solution with0.643 M TEA, pH 5.7, and osmolality 530 mmol/kg. The extrusion included10 passages through two stacked polycarbonate membranes with pore size100 nm. The resulting TEA-Pn and TEA-SOS liposomes had the size of112.3±15.5 nm and 120.5±42.5 nm, respectively (mean±SD of the sizedistribution). The liposomes were loaded with CPT-11 at the inputdrug/phospholipids ratio of 500 mg/mmol. The resulting liposomes had thedrug content of 465.6±26.5 (93% loading efficiency) and 499.9±22.5 mg(100% loading efficiency) of CPT-11/mmol phospholipid for the TEA-Pn andTEA-SOS formulations, respectively.

HT-29 cells were obtained from American Type Culture Collection,Rockville, Md., and propagated in DMEM medium supplemented with 10%fetal calf serum, 50 U/ml penicillin G, and 50 μg/mL of streptomycinsulfate at 37° C., 5% CO₂ as recommended by the supplier. NCR nu/nuhomozygous athymic male nude mice (6 week old, weight at least 16 g)were obtained from Charles River. The mice were inoculatedsubcutaneously in the right flank with 0.1 mL of the suspensioncontaining 5×10⁶ cells suspended in the growth medium withoutantibiotics. Eleven days later the animals having tumors with the sizebetween 150 mm³ and 350 mm³ were assigned to the treatment groupsaccording to the following method. The animals were ranked according tothe tumor size, and divided into 6 categories of decreasing tumor size.Six treatment groups of 11 animals/group were formed by randomlyselecting one animal from each size category, so that in each treatmentgroup all tumor sizes were equally represented. Starting at day 13, theanimals received four tail vein injections, at the intervals of 4 days,of the following preparations: 1) Control (HEPES-buffered saline pH6.5); 2) Free CPT-11 50 mg/kg, administered as freshly prepared 5 mg/mLsolution in unbuffered physiological saline; 3) TEA-Pn liposomal CPT-11at 25 mg/kg per injection; 4) TEA-Pn liposomal CPT-11 at 50 mg/kg perinjection; 5) TEA-SOS liposomal CPT-11 at 25 mg/kg per injection; 6)TEA-SOS liposomal CPT-11 at 50 mg/kg per injection. The animal weightand tumor size were monitored twice weekly as described in Example 10.The weight of tumor was subtracted from the animal weighing results toobtain animal body weight. The animals were observed for 60 daysfollowing tumor inoculation. When the tumors in the group reached 20% ofthe mouse body weight, the animals in the group were euthanized. Therewere complete tumor regressions in some groups without the signs oftumor regrowth at the end of study. The tissues from the tumorinoculation site from these animals were collected and preserved forpathological analysis for residual tumor cells.

The results of this study are shown in FIGS. 6 and 7. Free CPT-11 hadonly minor effect on the tumor growth. All liposomes had pronouncedeffect resulting in tumor regression later followed by regrowth in mostanimals. 50 mg/kg dose was more effective than 25 mg/kg dose in bothTEA-Pn and TEA-SOS CPT-11 liposomes. Average tumor doubling timescalculated from the tumor size data (FIG. 7) were: control—4.2 days;free drug, 50 mg/kg—4.8 days; TEA-Pn liposomal drug, 25 mg/mg—43.6 days;TEA-Pn liposomal drug, 50 mg/kg—47.5 days; TEA-SOS liposomal drug at 25mg/kg—48.2 days, and TEA-SOS liposomal drug at 50 mg/kg—over 56 days(doubling time was not reached). Thus, liposomal CPT-11 preparedaccording to the present invention was at least about 10-fold moreactive than the free drug, given at the same dose and schedule.Unexpectedly, TEA-SOS CPT-11 liposomes were prominently more effectivein reducing tumor growth than TEA-Pn CPT-11 liposomes administered atthe same dose. While in the groups treated with free drug and TES-Pnliposomal drug at 50 mg/kg per injection there were no animals withouttumor regrowth, in the groups receiving 25 mg/kg of each liposomalformulation, one animal (9.1%) was tumor-free at the end of study, andin the group receiving 50 mg/kg of TEA-SOS liposomal CPT-11 formulation,at the end of study 4 animals (36.4%) were tumor-free without signs ofregrowth.

The drug manifested some toxicity. The animals receiving free CPT-11,but not liposomal CPT-11, experienced temporary morbidity (loss ofalertness, humped posture, ruffled fur, decreased mobility) for aboutone hour after drug injection. The animals receiving free CPT-11suffered permanent loss of about 6% of weight during treatment, and didnot recover, The animals receiving both liposomal CPT-11 formulationsexperienced transient weight loss at the time between second and thirdinjections averaging about 5% (at 25 mg/kg) or about 9% (at 50 mg/kg) ofthe pre-treatment value, and eventually attained normal weight.Therefore, the toxicity of liposomal drug was not more than that of thefree (non-liposomal) drug, while the efficacy of the liposomal drug wassubstantially higher. The weight loss was reversed when the drugtreatment was finished, and all animals recovered their weight withoutterminal morbidity or toxic deaths. Later on, the animals gained weightconcomitantly with tumor regressions. In the saline control group,animals that developed large tumors experienced weight loss evidentlydue to tumor-related morbidity. Overall, the liposome drug formulationwhere the drug was loaded into the liposomes having pre-entrappedpolyanionized sugar (sucrose octasulfate) proved to be the mostefficacious while having less toxicity than the non-liposomal drug.

Example 16. Toxicity of Free and Liposomal CPT-11 in Mice

Acute toxicities of free CPT-11 and liposome-encapsulated CPT-11prepared according to the present invention were compared by determiningthe maximum tolerated dose (MTD) following single i.v. injection inregular (immunocompetent) mice.

The following materials were used:

1) CPT-11 (Irinotecan Hydrochloride) preparation having IrinotecanHydrochloride 98.9% by HPLC, and moisture 7.6%. In this study drugformulations were prepared on the “as is” basis, without correction forthe moisture content or the Irinotecan base content.

2) Liposomal CPT-11 (Ls-CPT-11) was prepared as in Example 11, usinglipid matrix of DSPC 200 mol. parts, Cholesterol 133 mol. parts,PEG-DSPE 1 mol. part; entrapped solution TEA-SOS having 0.65 M TEA, pH6.4; drug loaded into liposomes in 5 mM HEPES buffer, 5% dextrose, pH6.5, at 60° C. for 30 min at the input drug/lipid ratio 500 mg drug/mmolof phospholipid. Loading efficiency was >99%. Liposome size (volumeaverage mean±standard deviation by QELS): 101±37 nm. Liposomes wereformulated in the vehicle, 20 mM HEPES-Na, 135 mM NaCl; pH 6.5. Drugconcentrations in the injected formulations were as stated in the Tablesbelow.

3) Free CPT-11 solution. Free drug stock solution was prepared bydissolving Irinotecan Hydrochloride in 5% aqueous dextrose at 22 mg/mL,and sterilized by 0.2-μm filtration. This stock solution was dilutedwith sterile 5% dextrose prior to injection.

4) Animals. Female Swiss Webster mice, 6-8 week old, were from Harlan,USA.

MTD determination generally followed the protocol adopted by the UnitedStates National Cancer Institute Developmental Therapeutics Program. Theprotocol included the following three steps:

Step 1): Range-seeking step with the dose escalation factor of 1.8. Thegroups of two animals were injected into the tail vein with increasingdoses of the free or liposomal Irinotecan, beginning with the dose of 60mg/kg, and continuing with the dose escalation factor of 1.8, untilacute mortality or terminal morbidity (within >1 day post injection) isobserved in any of the animals. The dose one step below themortality/terminal morbidity dose is recorded.

Step 2): Range-seeking step with the dose escalation factor of 1.15. Thegroups of two animals were injected into the tail vein with increasingdoses of the free or liposomal Irinotecan, beginning with the doserecorded at Step 1, and continuing with the dose escalation factor of1.15, until acute mortality or terminal morbidity (within >1 day postinjection) is observed in any of the animals. The dose one step belowthe mortality/terminal morbidity dose is recorded at tentative MTD.

Step 3): Validation step. The group of 5 animals is injected i.v. (tailvein) with free or liposomal Irinotecan at tentative MTD determined atStep 2. The animals are followed for 7 days, the animal body weight isrecorded twice weekly and compared with the pre-injection weight.General health of the animals is observed (alertness, grooming, feeding,excreta, skin, fur, and mucous membrane conditions, ambulation,breathing, posture). If during the observation period there is nomortality, progressive morbidity, or weight loss in excess of 15% of thepre-injection body weight, the dose is considered to be validated asacute single injection MTD. If any of these effects occur, theexperiment is repeated at the next lower dose by a factor 1.15.

To obtain additional statistics for validation step, the body weightdynamics of surviving animals was followed for up to 11 days postinjection. The dose of more than 324 mg/kg of the liposomal Irinotecanwas impossible to administer because of the concentration and injectionvolume limitations. The results are presented in Table 10.

TABLE 10 MTD seeking study of CPT-11 formulations in mice. Step 1.increase dose by a factor of 1.8 Animal body weight, at day postinjection: inj. Dose drug conc. Inj. volume mouse 0 1 2 4 5 6 7 11 drug(mg/kg) (mg/ml) μl # (g) (g) (g) (g) (g) (g) (g) (g) La-CPT11 60 8 150 119.2 18.0 nd 20.3 20.6 20.6 20.0 19.7 2 19.7 19.3 nd 20.6 20.4 19.6 19.720.7 100 12 165 1 19.5 18.6 nd 19.6 20.0 20.1 19.4 19.9 2 20.1 18.9 nd20.2 21.5 22.2 21.8 22.5 180 22 165 1 19.4 18.4 nd 18.9 19.7 20.5 19.520.5 2 20.0 19.3 nd 19.6 20.6 21.4 21.6 21.7 324 30.6 210 1 21.8 21.221.2 nd 20.2 nd 20.2 nd 2 21.6 20.4 21.3 nd 20.8 nd 21.4 nd free CPT1160 8 150 1 20.6 20.4 nd 22.1 22.1 22.2 22.0 22.5 2 19.5 19.1 nd 20.220.3 20.4 20.5 21.1 100 12 165 1 19.3 died 1-2 min after injection 220.1 died 1-2 min after injection 3 19.9 died 1-2 min after injectionStep 2. increase dose by a factor of 1.15 Animal body weight, at daypost injection: inj. Dose drug conc. Inj. volume mouse 0 1 2 5 7 drug(mg/kg) (mg/ml) μl # (g) (g) (g) (g) (g) free CPT11 60 8 150 3 19.9 20.020.9 19.9 21.3 4 19.5 18.7 19.4 18.8 18.9 70 8 175 5 20.9 20.0 20.6 19.320.4 6 22.3 21.8 22.4 22.4 22.8 80 8 200 7 20.6 19.9 20.1 19.9 20.9 820.6 20.8 21.1 20.7 21.4 90 12 150 9 22.3 died 1-2 min after injection10 22.4 died 1-2 min after injection 8 225 11 20.6 died 1-2 min afterinjection Step 3. Validation Animal body weight, at day post injection:inj. Dose drug conc. Inj. volume mouse 0 3 5 7 drug (mg/kg) (mg/ml) μl #(g) (g) (g) (g) Free CPT11 80 8 200 1 20.2 19.3 20.0 21.7 2 20.5 20.620.5 21.2 3 20.7 20.6 20.8 21.9 4 20.8 21.4 22.1 23.0 5 21.9 21.9 21.621.5 Ls-CPT11 324 36.5 180 6 21.0 20.0 20.1 20.2 7 20.4 20.4 20.2 19.2 820.4 19.8 20.3 20.7 9 20.9 19.9 20.5 21.5 10 20.7 19.5 19.8 20.2 Afterinjection, all mice treated with free CPT11 were sick, short of breathfor about 1 h and then recovered After injection, all mice treated withLs-CPT11 were normal

Thus, while the MTD of free CPT-11 was 80 mg/kg, the MTD of liposomalCPT-11, surprisingly, was not achieved even at the highest administereddose of 324 mg/kg. Therefore, liposome encapsulation of CPT-11 accordingto the present invention has reduced the drug toxicity at least 4-fold.

Example 17. Storage Stability of CPT-11-Loaded TEA-SOS Liposomes AgainstDrug Leakage

Five batches of liposomal CPT-11 were prepared using the TEA-SOS method(Example 11), at the drug/lipid input ratio of 500-550 mg/mmolphospholipid. The liposomes were prepared using membrane extrusionthrough polycarbonate membrane with 80 nm or 100 nm pore size, asindicated in the table below. The liposomes were 0.2-μm filtersterilized and stored at 3.4-14.5 mg/mL of CPT-11 in 135 mM NaCl, 20 mMHEPES-Na, pH 6.5 (storage buffer), at 4-8° C. After the indicatedstorage time, the leaked drug was removed by gel-chromatography onSephadex G-75 using the storage buffer as eluent. The drug andphospholipid concentrations in the liposomes before and aftergel-chromatography were assayed using spectrophotometry method and aciddigestion-blue phosphomolybdate method, respectively, as described inExamples 70 and 71. CPT-11 liposomes prepared according to the presentinvention were very stable. The leakage of CPT-11 from these liposomesduring storage was less than 5% over 6 months (Table 10).

TABLE 11 Encapsulation stability of CPT-11 liposomes during storage(data are mean ± SE). Extrusion CPT-11 Storage % drug Liposome poresize, concentration, time, remaining Lot # nm mg/ml months encapsulated1 80 3.44 ± 0.06 6 99.02 ± 3.77 2 80 7.88 ± 0.19 6 102.38 ± 4.78  3 1004.57 ± 0.06 6 96.38 ± 4.69 4 100 4.62 ± 0.11 6 95.72 ± 4.36 5 80 14.52 ±0.42  3 103.4 ± 5.92

Example 18. Liposomes Loaded with Topotecan

Liposomes with entrapped TEA-Pn solution and TEA-SOS solution wereprepared as in Example 11. Stock solution of Topotecan Hydrochloride(GlaxoSmithKline, Pa., USA) was prepared immediately before mixing withthe liposomes by dissolving Topotecan Hydrochloride in water at 15-20mg/ml, counting on the actual Topotecan HCl content. The pH was adjustedto 3.0 with 1 N HCl. The drug solution was filtered through 0.2 micronpolyethersulfone (PES) sterile filter using positive pressure. Aliquotsof the TEA-Pn or TEA-SOS-containing liposomes in the drug-loading bufferwere mixed at room temperature with the stock Topotecan HCl solution toachieve the drug/lipid input ratio in the range of 0.15-0.45 g/mmol ofliposome phospholipid. Preferred ratio was 0.35 g of Topotecan HCl formmol of liposome phospholipid. The mixtures in glass containers wereincubated on the thermostatted water bath at 55-62° C. with slowagitation for 30-60 min, quickly cooled down in ice-water bath (0-2° C.)and left at this temperature for 5-15 min. This step resulted in theencapsulation efficiency of 89-90% (TEA-Pn gradient) or 97-100% (TEA-SOSgradient). Unencapsulated Topotecan was removed, and the liposomes weretransferred into the storage buffer using size exclusion columnchromatography. Before application on the column, the ionic strength ofthe liposome preparation was increased by mixing with 1/20 vol. of 2.88M aqueous sodium chloride, and the mixture was incubated for about 15min. We unexpectedly found that adjusting the ionic strength of theliposome medium from the low value during the loading (typicallyequivalent to less than 20 mM NaCl) to the higher value of above 20 mMNaCl, and preferably to 50 mM NaCl and above, improved the removal ofunencapsulated drug and increased the stability of Topotecan-loadedliposomes against aggregation, possibly by facilitating the removal ofmembrane-bound Topotecan, as opposed to the drug encapsulated in theliposome interior. The rest of the procedure followed Example 11, step7. For the results, see Table 12 below.

Example 19. Preparation of Anti-HER2-Immunoliposomal Formulations ofTopotecan

Topotecan immunoliposomes specifically internalizable by cancer cellsoverexpressing HER2 (C-ErbB-2) surface receptor tyrosine kinaseoncoprotein were prepared by conjugating Topotecan liposomes toanti-HER2 single chain human Fv antibody fragment, F5, selected from thephage display library for its high internalization intoHER2-overexpressing cells (Poul, et al., 2000, J. Molecular Biology, v.301, p. 1149-1161). F5 is a 27-KDa protein that binds to extracellulardomain of HER2 receptor with affinity of about 150 nM, causing rapidinternalization (Neve, et al., 2001, Biophys. Biochim. Res. Commun. v.280, p. 274-279). For liposome conjugation, the method of U.S. Pat. No.6,210,707 and of Nielsen, et al. (2002), Biochim. Biophys. Acta, v.1591, p. 109-118, were generally followed. A hydrophilic lipopolymerconjugate of F5 was first prepared. C-terminus of F5 amino acid chainhad an added terminal terminal cysteine group (F5Cys). The F5Cysconstruct was expressed in E. coli and isolated from the bacteriallysate by Protein A column chromatography. Protein A eluted fractionswere adsorbed on anion-exchange resin to remove pyrogens and host DNA,and treated with a thiol reducing agent to liberate the thiol group ofthe terminal cysteine. The reduced F5Cys was further purified by ionexchange chromatography using SP Sepharose Fast Flow (AmershamPharmacia). The purified protein was conjugated to a thiol-reactivelipid-poly(ethylene glycol) linker,N-(3-(N-maleimido)propyonylamido)-poly(oxyethylene)-oxycarbonyl)-1,2-distearoylphosphatidylethanolamine (Mal-PEG-DSPE), a derivative of PEG with mol. weight 2,000,commercially produced by Avanti Polar Lipids, Inc., Alabama, USA. Theprotein and the linker were incubated in aqueous buffer solution at themolar ratio of 1:4, and the un-reacted linker was quenched with 1 mMcysteine. During the reaction, terminal cysteine of F5Cys is covalentlyattached to maleimido group of the linker. The resulting F5-PEG-DSPEconjugate was water soluble in the form of micelles having high apparentmolecular weight (500-850 KDa), and was separated from unreacted protein(about 25%) by size exclusion chromatography. The amount of protein inthe purified conjugate was determined by UV spectrophotometry at 280 nm,and the amount of the linker was assayed using a spectrophotometricmethod identical to that used for phospholipid quantification (seeExample 70) The purified F5-PEG-DSPE conjugate was stable in water,fully immunoreactive, and was stable against denaturation and loss ofreactivity for at least 1 hour at 65° C. and at least 3 months at 37° C.

To prepare anti-HER2 immunoliposomal Topotecan, Topotecan-loadedliposomes of Example 18 were mixed with F5-PEG-DSPE in the aqueoussaline buffer at the ratio of 15 microgram of protein per 1 micromole ofphospholipid (about 45 F5 copies per liposome). The mixture wasincubated for 40 min. at 60° C., chilled on ice, and chromatographed ona column with Sepharose CL-4B (cross-linked 4% agarose beads, AmershamPharmacia) to remove residual micellar conjugate, unconjugated protein,and any traces of extraliposomal drug that may have been released duringthe incubation. The liposomes with membrane-incorporated F5-PEG-DSPEwere eluted with 5 mM HEPES-144 mM NaCl buffer pH 7.4, collected in thevoid volume of the column, sterile-filtered and dispensed for storage(4-6° C.). The amount of liposome-incorporated F5 was typically >80% ofthe added conjugate. It was determined by SDS-PAGE of the liposomes withquantification of the Coomassie-stained F5 band by densitometry. Drugand lipid concentrations in the immunoliposome preparations weredetermined similarly to non-targeted liposomes. The properties ofTopotecan liposomes and F5-immunoliposomes (Examples 18-19) aresummarized in Table 12.

TABLE 12 Characteristics of Topotecan liposomes and immunoliposomes.Liposome- F5 scFv Drug/lipid ratio, % Liposome entrapped attach- g/molphospholipid encap- size, Mean ± salt ment: Input Output sulation SD, nmTEA-Pn No 173.6 155.2 ± 5.9 89.4 ± 3.4% 96.4 ± 38.7 TEA-Pn Yes 173.6156.2 ± 5.2 90.0 ± 3.0% 96.2 ± 33.8 TEA-SOS No 347.2  340.8 ± 14.7 98.2± 4.2% 99.1 ± 32.6

Example 20. Effect of Loading Buffer pH and Drug/Lipid Ratio on theTopotecan Loading into Liposomes

Liposomes (DSPC/Chol/PEG-DSPE, 3:2:0.015 molar ratio) with entrapped 0.5N TEA-Pn, pH 6.2, osmolality 413 mmol/kg, were prepared using theethanol injection method (Example 18), extruded through two stackedpolycarbonate filters with 100 nm pore size 5 times and with 50 nm poresize 10 times. The loading buffer was 5 mM MES, 50 g/L Dextrose,adjusted to various pHs in the range 5.0-6.5. The liposome size was73.1±21.3 nm by QELS. The liposomes were loaded by mixing a Topotecanstock solution (20 mg/ml) with the liposomes in the loading buffer atthe input drug-to-phospholipid ratio of 100 mg/mmol, incubating themixture at 60° C. for 45 min, quenching on ice for 15 min and removingthe unencapsulated drug using a Sephadex G-75 column eluted with 20 mMHEPES, 135 mM NaCl, pH 6.5. Topotecan and phospholipid were quantifiedby spectrophotometry (Examples 70 and 71). The results (Table 13)indicated that Topotecan loading was nearly quantitative in the range ofpH 5.5-6.5.

TABLE 13 Effect of loading buffer pH on the % of Topotecan encapsulationinto the liposomes with entrapped TEA-Pn. Loading buffer pH %encapsulation 5.0 50.1 ± 2.1 5.5 97.2 ± 8.1 6.0 115.5 ± 15.0 6.5 102.1 ±8.1 

The effect of drug to lipid ratio (0.15-0.45 mg/mmol phospholipid) onthe loading efficiency was also studied. The liposomes with entrappedTEA-Pn (0.5 M TEA, pH 5.8, osmolality 480 mmol/kg) were prepared asabove, except the final extrusion step was ten times through two stacked0.08 μm polycarbonate filters. The loading was at pH 6.5. The liposomesize was 93.1±15.1 nm by QELS. The results (Table 14) showed that drugloading efficiency was over 85% over the whole range of drug/lipidratios studied.

TABLE 14 Effect of drug/lipid ratio on the encapsulation efficiency ofTopotecan into the liposomes containing TEA-Pn. Topotecan/phospholipidratio, mg/mmol % encapsulation Input ratio Output ratio (after loading)(mean ± SE) 168.2 166.9 ± 11.1 99.2 ± 6.6 224.4 232.5 ± 47.6 103.7 ±21.2 280.3 253.5 ± 19.8 90.4 7.0 336.4 298.3 ± 18.0 88.7 ± 5.3 392.5361.2 ± 36.8 92.0 ± 9.4 448.5 394.9 ± 29.5 88.0 ± 6.6

Example 21. Topotecan Liposome Stability In Vitro in the Presence ofPlasma

Liposomes (DSPC/Chol/PEG-DSPE, molar ratio 3:2:0.015) with entrapped 0.5N TEA-Pn, pH 6.2, osmolality 413 mmol/kg, were prepared as described inExample 18. Liposomes with the size of 96.4±29.3 nm were produced byextrusion ten times through two stacked 100 nm pore size polycarbonatefilters. For quantitation of the liposome lipid in plasma, [³H]-CHE wasincluded in the lipid solution at 0.5 μCi/μmol of DSPC. Topotecan wasloaded at pH 6.0, 58° C. for 45 min at a drug/phospholipid ratio of 150mg/mmol. The efficiency of loading was 148.48±10.26 μg Topotecan/μmolphospholipid (99.0±6.8%).

The liposomes were incubated with 50% human plasma in a multiwellmicrodialysis device (Spectra-Por MicroDialyzer 10-well, Spectrum, USA).Human donor plasma was diluted by the equal volume of HEPES-bufferedsaline (20 mM HEPES, 135 mM NaCl), pH 6.5, containing 0.02% sodium azideand charged into the lower reservoir of the dialyzer (32 mL). The wells(0.4 mL) were separated from the reservoir by a polycarbonate membranewith 30 nm pore size, to afford free passage of plasma proteins andsmall molecules but not the liposomes. The liposomes were mixed withcalculated amounts of plasma and HEPES-buffered saline to achieve theconcentration of 2.5 mM phospholipid and 50 vol. % of plasma. The devicewas incubated at 37° C., and the contents of the reservoir were stirredslowly. After 8 hours of incubation, the contents of the lower reservoirwere changed for fresh 50% plasma. At the given time points (see below)50-μL aliquots were withdrawn from the wells, and chromatographed on thecolumns containing 2.2-2.4 mL of Sepharose CL-2B, eluent HEPES-bufferedsaline to separate the liposomes from plasma proteins and free drug. Theliposomes were collected in the void volume fractions. Topotecan wasquantified by fluorometry using excitation at 384 nm and emission at 524nm after solubilization of the plasma samples in 90% aqueousisopropanol-0.1 N HCl, and the lipid was quantified by scintillationcounting of [³H]-CHE (quenching corrected). The determined drug-to-lipidratio at time was compared to the initial ratio prior to incubation toobtain the % of Topotecan that remained encapsulated at each time point.After 8 hours of incubation, the amount of drug remaining in theliposome was about 55% of its initial value (Table 15).

TABLE 15 In vitro release of Topotecan from the liposomes loaded byTEA-Pn gradient in 50% human plasma at 37° C. Incubation time, hours %drug remaining encapsulated 1 95.5 ± 5.4 4 76.8 ± 7.3 8 55.9 ± 4.1 24 55.4 ± 16.8

Example 22. Topotecan Liposomes with Entrapped TEA-Pn Gradient atVarious Drug/Lipid Ratios: In Vivo Drug Retention and CirculationLongevity in Mice

The liposomes (DSPC/Chol/PEG-DSPE at 3:2:0.015 molar ratio, containing[³H]-CHE at 0.5 mCi/mmol DSPC) with encapsulated gradient-forming saltsolution (0.5 N TEA-Pn, pH 6.2, osmolality 413 mmol/kg) were prepared asin Example 18 using extrusion 12 times through two stacked 100 nm poresize polycarbonate filters. The liposome size was 107.7±19.1 nm by QELS.The liposomes in 5 mM HEPES, 50 g/L Dextrose, pH 6.5 were mixed with theaqueous stock solution of Topotecan (20 mg/ml) at drug/phospholipidratios in the range 130-360 μg/μmol, followed my incubating the mixtureat 58° C. for 45 min, placing on ice for 15 min and removal ofunencapsulated drug by Sephadex G-75 chromatography. Twelve-week oldfemale FvB mice were injected with the liposomes via the tail vein at adose of 5 mg Topotecan per kg body weight (approx. 0.2 mgTopotecan/animal) in triplicate. At indicated times, typically 8 hoursor 24 hours post injection, the mice were anesthetized, exsanguinated,and the blood samples were assayed for the drug and the liposome lipidas in Example 8. The results are shown in Table 16. After 24 hours,about 6-32% of the initial drug load remained encapsulated. Higher loadsof the drug (>200 mg/mmol phospholipid) resulted in longer drugretention.

TABLE 16 In vivo drug retention and circulation longevity of prototypeTopotecan liposomes loaded using TEA-Pn gradient method to differentdrug/lipid ratios. Lipid remaining Topotecan remaining in circulation,encapsulated, Encapsulated % injected dose % of initial loaddrug/phospholipid After 8 After 24 After 8 After 24 ratio, mg/mmol hourshours hours hours 127.2 ± 10.9 36.1 ± 2.0 18.7 ± 8.1  51.7 ± 7.1 6.72 ±2.5 207.2 ± 21.6 32.1 ± 5.2 9.84 ± 1.88  75.6 ± 13.0 13.8 ± 3.5 301.3 ±24.5 34.4 ± 3.2 8.04 ± 4.25 79.2 ± 4.2 25.6 ± 4.4 360.3 ± 35.6 33.6 ±2.4 8.68 ± 4.96 73.5 ± 7.0 32.3 ± 9.8

Example 23. In Vivo Drug Retention and Circulation Longevity ofTopotecan Liposomes Loaded Using Different Entrapped Ammonium andTriethylammonium Salts

The liposomes composed of DSPE, cholesterol, and PEG-DSPE (3:1:0.1 byweight), also containing [³H]-CHE at 0.22 mCi/mmol DSPE, were preparedas in Example 18, except that the extrusion step included 10 passagesthrough 2 stacked 200-nm pore filters, 10 passages through 2 stacked100-nm pore filters, and 20 passages through 2 stacked 50-nm porefilters. The liposomes contained the following salt solutions:

0.5 N ammonium dextran sulfate solution (A-DS) was prepared from sodiumdextran sulfate (M.w. 5000), purchased from Sigma, and converted intoammonium salt by the ion exchange procedure similar to that of Example4. The solution of dextran sulfuric acid was immediately titrated with12.4 M aqueous ammonia. The A-DS solution has pH 5.66, osmolality 208mmol/kg.

0.48 N ammonium sucrose octasulfate (A-SOS) was prepared similar toExample 6, but ammonium hydroxide was used for titration. The solutionhad pH 6.27, osmolality 258 mmol/kg.

0.47 M triethylammonium sucrose octasulfate (TEA-SOS) was prepared as inExample 6. The solution has pH 6.6, osmolality 297 mmol/kg.

Topotecan was loaded into the liposomes in the aqueous solution of 10 mMMES-Na, 50 g/L dextrose, pH 6.5, by incubating the liposomes with thedrug at 61-62° C. and input drug/phospholipid ratio of 346±1 mg/mmol,for 40 min, followed by incubating on ice for 10 min. The liposomes werepurified from unencapsulated drug by chromatography on Sephadex G-25,eluent—aqueous 2 mM Histidine, 144 mM NaCl, pH 6.6 (HCl).

Seven to nine week old female Swiss Webster mice were injected via thetail vein with these liposomal Topotecan formulations at the dose of 5mg Topotecan per kg body weight (approx. 0.2 mg Topotecan/animal) intriplicate. After 8 hours or 24 hours post injection the blood wascollected and analyzed for Topotecan and liposome lipid as in Example22.

The results are presented in Table 17 below. While all three liposomeformulations demonstrated very close liposome circulation longevity,having about 23-28% of the injected dose remaining in blood 24 hourspost injection, unexpectedly the drug retention in TEA-SOS liposomes andin A-SOS liposomes was better than in A-DS liposomes both in terms ofmagnitude (about 2-fold improvement in drug retention) and statisticalsignificance (statistical significance at 95% confidence level by2-tailed non-paired Student's t-test p=0.0257 and p=0.00995,respectively; and by Mann's U-test the difference was significant withα=0.01). Drug retention in TEA-SOS containing Topotecan liposomes wasbetter than in A-SOS containing Topotecan liposomes.

TABLE 17 In vivo drug retention and circulation persistence of Topotecanliposomes prepared using TEA-SOS, ammonium-SOS (A-SOS), and ammoniumdextran sulfate (A-DS. Lipid remaining in Topotecan remainingcirculation, % encapsulated, % Drug/phospho- Loading injected dose ofinitial load lipid ratio, efficiency, Liposome After 8 After 24 After 8After 24 Gradient mg/mmol % size, nm hours hours hours hours A-DS 288.1± 20.6 83.3 ± 6.0 76.9 ± 22.7 43.7 ± 1.2 27.7 ± 1.5 43.6 ± 6.8 18.7 ±1.5 A-SOS 346.2 ± 14.3 100.0 ± 4.1  99.7 ± 28.9 42.3 ± 2.2 23.4 ± 2.053.3 ± 0.8 31.3 ± 3.2 TEA-SOS 340.8 ± 14.7 98.5 ± 4.2 99.1 ± 32.6 42.1 ±2.3 23.0 ± 2.9 57.0 ± 5.6 38.1 ± 6.1

Example 24. Drug and Lipid Plasma Pharmacokinetics of LiposomalTopotecan in Rats

The circulation longevity and Topotecan release parameters were assessedin rats. The liposomes (DSPC/Cholesterol/PEG-DSPE molar ratio 3:2:0.015)were prepared by ethanol mixing/extrusion method and loaded withTopotecan using TEA-Pn gradient or TEA-Sucrose octasulfate (TEA-SOS)gradient as described in Example 18 and loaded at various drug/lipidratios (15-450 mg/mmol phospholipid). For lipid matrix quantification,the liposome lipid contained [3H]-CHE at 0.5-1.5 mCi/mmol DSPC. FemaleSprague Dawley rats (6-8 week old; body weight about 200 g) withindwelling central venous catheters were injected i.v. (via thecatheter) with the Topotecan liposomes at the dose of 4-5 mg/kg of bodyweight. The catheter was flushed with saline. At selected times (up to48 hours post injection) the blood samples (0.2-0.3 mL) were drawn viathe catheter into heparinized syringes, mixed with 0.4 mL of coldphosphate buffered saline with 0.04% EDTA, blood cells were separated bycentrifugation, and the supernatants (PBS-diluted plasma) were assayedfor lipid by ³H-CHE radioactivity counting (quenching corrected), andfor Topotecan by fluorometry (Example 71). The assay results werecorrected for plasma dilution, calculated from the weight of obtainedblood sample and assuming a hematocryt of 40%. The total blood dose ofthe drug and lipid was estimated from the blood volume calculated as6.5% of the body weight. The percent of Topotecan retained in theliposomes was calculated by comparing the drug/lipid ratio at a giventime point to the drug/lipid ratio of the injected liposomes. Table 18below summarizes blood half-lives of the lipid, the drug, and thehalf-lives for drug release, as well as other properties of theliposomes. Pharmacokinetic (PK) curves are shown on FIGS. 8A (lipid) and8B (drug/lipid ratio). In summary, the blood PK curves for both drug andlipid fit well to single exponent model (R² 0.984-0.999). Despite their90-100 nm size and very small amount of PEGylated lipid (0.3 mol. %),the liposomes unexpectedly showed good circulation longevity (plasmahalf-lives of the lipid component were in the range of 11-16 hours). Theslowest release of Topotecan (half-time 22.9 hours) was observed withthe liposomes loaded using the TEA-SOS method.

TABLE 18 Circulation half-life (t_(1/2)) of lipid, drug, and half-timefor drug release from the prototype Topotecan liposomes in rats.Topotecan load, Liposome Injected t_(1/2) t_(1/2) t_(1/2) of drug No. ofEntrapped salt, mg/mmol size, nm dose, lipid, drug, release, animals andconcentration phospholipid (mean ± SD) mg/kg hours hours hours per groupTEA-Pn 0.5N 124.3 ± 9.7   92.3 ± 23.3 4 15.8 4.13 5.34 3 TEA-Pn 0.5N360.3 ± 35.6 107.7 ± 19.1 5 12.8 6.06 9.97 2 TEA-SOS 0.643N 439.2 ± 15.9108.8 ± 13.4 5 10.8 7.36 22.87 2

Example 25. Drug Stability Against Leakage During Storage of TopotecanLiposomes

The samples of several prototype formulations prepared for theabove-described studies, were stored at 4-6° C. for various times toassess the storage stability of the encapsulated Topotecan against drugleakage from the liposomes. The liposome samples were passed throughSephadex G-75 columns, eluted with 20 mM HEPES, 135 mM NaCl, pH 6.5, toremove extraliposomal drug, and analyzed for drug content byspectrophotometry and for lipid by [3H]-CHE radioactivity counting. Theresults (Table 19) indicate good retention of Topotecan in the liposomesduring storage.

TABLE 19 Drug retention in prototype Topotecan liposomes during storage.Initial Drug load Liposome Liposome size, drug load, Storage afterstorage gradient- mean ± SD, mg drug/mmol time, as % of forming salt nmphospholipid months initial TEA-Pn  96.4 ± 29.3 148.5 ± 10.3 8 101.6 ±5.5 0.500N pH 6.2 TEA-Pn 107.7 ± 19.1 127.2 ± 10.9 6  94.6 ± 6.2 0.500NpH 6.2 TEA-Pn 107.7 ± 19.1 207.2 ± 21.6 6 113.9 ± 9.4 0.500N pH 6.2TEA-Pn 107.7 ± 19.1 301.3 ± 24.5 6 112.9 ± 9.3 0.500N pH 6.2 TEA-SOS108.8 ± 13.4 439.2 ± 15.9 2  97.8 ± 9.4 0.643N pH 5.6

Example 26. In Vitro Uptake of Liposomal and Immunoliposomal Topotecanby HER2-Overexpressing Cancer Cells

This study addressed the capacity of Topotecan-loadedanti-HER2-immunoliposomes prepared according to the invention to deliverTopotecan specifically into HER2-overexpressing cells in cell culture.The (immuno)liposomes were prepared and loaded with Topotecan usingTEA-Pn method of Example 19. HER-2 overexpressing human breast carcinomacells (SKBr-3, ATCC) were grown in modified McCoy 5A medium (withouttricine) supplemented with 10% fetal calf serum, 50 μg/mL streptomycinsulfate and 50 U/ml penicillin G (complete growth medium) in T-75 flasksat 37° C., 5% CO₂, to confluency. The cells were harvested bytrypsinization, inoculated into 24-well cell culture plates at 150,000cells/well in 0.5 mL of the complete growth medium, and allowed toacclimate overnight. The medium was replaced with 0.5 mL of completegrowth medium containing Topotecan formulations at the selectedconcentration in the range of 0.01-0.1 mM phospholipid. Triplicate wellswere used for each condition. Control wells were incubated in theabsence of drug and/or liposomes (to obtain background readings for drugassay). The plates were incubated with slow agitation at 37° C., 5% CO₂for 4-8 hours. The media were aspirated, and the cells were rinsed 4times with 1 mL portions of cold Hanks' balance salt solution containingCa and Mg salts. The cells were solubilized by adding 0.1 mL of 1%Triton X-100 in water, and the amount of drug in the cell lysates wasdetermined by fluorometry (Example 71). The standard curve was obtainedin the range of 10-2500 ng Topotecan/well, and fit to second orderpolynomial (to account for self-quenching at higher drug concentration)after subtracting the cell autofluorescence background. When amicroplate fluorometer was used, the filter selection was 400/30 nm forexcitation, 530/25 nm for emission. Both cuvette- and microplatefluorometers gave the same results.

The results of two experiments are summarized in Table 20 below. Therewas prominent cellular uptake of HER2-targeted liposomal drug (50-300times higher than of no-targeted liposomal Topotecan). Interestingly,uptake of free Topotecan was also significantly lower than ofHER2-targeted immunoliposomal Topotecan. This may be explained by rapidhydrolysis of the camptothecin lactone ring of Topotecan molecule in thecell growth medium in the presence of serum, generating the carboxylateform of the drug which may have lower cell permeability and lowercytotoxicity. In summary, the ability of cell-targeted, internalizable,ligand-conjugated immunoliposomes to deliver Topotecan intracellularlywas confirmed.

TABLE 20 In vitro cellular uptake of Topotecan liposomes and anti-HER2immunoliposomes containing TEA-Pn (nd, not determined). For liposomecharacteristics see Table 12. Liposome Topotecan Topotecan uptake bySK-Br-3 cells, ng/100,000 cells concentration, concentration ExposureNon-targeted F5-Immuno- Free mM phospholipid μg/mL time, hours liposomesliposomes drug 0.1 15.5 4 1.45 ± 0.09  163 ± 5.7 nd 0.01 1.55 4 0.185 ±0.03  60.2 ± 2.0 nd 0.033 5.0 8 3.62 ± 2.03 169.6 ± 13.7 5.56 ± 0.91

Example 27. Cytotoxicity of Liposomal and Immunoliposomal TopotecanAgainst HER2-Overexpressing Cancer Cells In Vitro

Once the capacity of anti-HER2 Topotecan immunoliposomes forintracellular drug delivery into HER2-overexpressing cancer cells wasestablished (Example 26), it was important to ensure that theinternalized liposomes can release the drug in its active form. To thisend, in vitro cytotoxicity of the free Topotecan (i.e., Topotecanformulated as a solution), liposomal Topotecan, andanti-HER2-immunoliposomal Topotecan was studied. The liposomal Topotecanformulations were prepared, and SKBr-3 cells were grown and harvested asdescribed in Example 26. The cells were inoculated into 96-well cellculture plates at 5,000 cells in 0.1 mL of the complete growth medium,in triplicate, and left to acclimate overnight. Edge-most rows andcolumns of the plate were left empty. Sterile preparations of Topotecanliposomes, immunoliposomes, or free drug (freshly prepared by dilutingTopotecan 20 mg/mL stock, pH 3, into unbuffered saline to 2 mg/mL) werediluted with complete drug medium to achieve concentrations startingfrom 90, 30, or 10 μg/mL and serially diluted down in the medium by thefactor of 3. The media in the wells were replaced with 0.2 mL ofdrug/liposome dilutions, and incubated at 37° C., 5% CO₂, for specifiedtime (4-6 hours). One well in each row was incubated with drug-freemedium to serve as a non-treated control. The drug-containing media wereaspirated from the wells, the cells were rinsed with 0.2 mL of drug-freemedium, and 0.2 mL of fresh drug-free medium was added to all wells. Theplates were incubated for 4 days at 37° C., 5% CO₂. Without mediumchange, 0.03 mL of the 2 mg/mL solution of a tetrazolium dye (ThiazolylBlue, MTT) (Sigma Chemical Co.) in serum-free medium was added to eachwell. The plates were incubated for additional 2-3 hours at 37° C., 5%CO₂. The media were aspirated, and the wells were filled with 0.2 mL of70 vol. % aqueous isopropanol, 0.075 N HCl, and agitated gently untilthe formazan dye dissolves (15-30 min). The optical density of theformazan solutions was determined using microplate photometer at 540 nm.The cell viability as % of non-treated control was calculated as theratio of the optical density in the experimental wells to the opticaldensity in the wells containing non-treated cells, corrected forbackground. The data were plotted against the drug concentration, andthe IC₅₀ dose was estimated graphically from intersection of theviability-concentration curve with the 50% viability line.

The results are presented in FIG. 9. The drug dose resulting in 50%growth inhibition (IC₅₀) for free Topotecan or non-targeted liposomalTopotecan was in excess of 30 μg/mL; for F5-Immunoliposomal Topotecan,0.15 μg/mL. These results are consistent with the targeted drug uptakedata.

Example 28. Comparative Stability and Plasma Pharmacokinetics ofLiposomal and F5-Immunoliposomal Topotecan in Mice

Topotecan liposomes containing radioactive lipid label [³H]-CHE at 1.5mCi/mmol of phospholipid were prepared according to Examples 11 and 19using an ethanol lipid solution mixing-extrusion procedure under thefollowing conditions: gradient-forming salt solution: 0.643 Ntriethylammonium sucrose octasulfate; polycarbonate membrane extrusion:15 passages through 2 stacked PCTE filters, 80 nm pore size; Topotecanloading: drug/phospholipid input ratio 350 mg/mmol (calculated forTopotecan free base); F5 scFv conjugation was performed as described inExample 19. The liposomes had the following characteristics:

Size by QELS: weight average 101.2 nm; standard deviation, 20.1 nm.

Drug encapsulation: Topotecan liposomes (Topo-Ls) 359.3±27.4 mg/mmolphospholipid; Topotecan F5scFv-immunoliposomes (Topo-F5-ILs) 326.3±15.9mg/mmol phospholipid.

The study was performed generally as in Example 22. The groups of ninemale Swiss Webster mice (8-10 week old, 24-27 g) were injected via tailvein with Topo-Ls, Topo-F5ILs, or freshly prepared Topotecan 1 mg/mL inunbuffered saline, at the dose of 5 mg Topotecan base per kg of the bodyweight (equivalent to the lipid dose of 14-16 μmol of phospholipid/kgbody weight). At 1 hour, 8 hour, or 24 hour post injection time points,3 animals per time point were exsanguinated via open heart punctionunder Ketamine/Xylazine anesthesia, the blood was collected into tubescontaining PBS-EDTA, and assayed for Topotecan (fluorometry) andliposome lipid (by radioactivity scintillation counting). The amounts ofdrug and lipid dose remaining in the blood at given time points werecalculated from the administered dose being taken as 100%, assuming theblood amount per animal as 6.3% of the body weight, and packed bloodcell fraction of 45%. The amount of drug remaining encapsulated in theliposomes at each time point was calculated for each animal individuallyby comparing drug/lipid radioactivity ratio of the plasma samples withthat of the injected liposomes. The amount of free Topotecan in theplasma samples collected at 1 hour post injection was less than 1% ofthe injected dose (indeed, they were below the detection limit of ourassay method); therefore, further time points of the free Topotecangroup were not studied. Because of the fast blood clearance and lowblood levels of free Topotecan we assumed that essentially all Topotecanfound in the blood at all time points represents liposomallyencapsulated Topotecan.

The results are summarized in Table 21 below. Remarkably, the liposomesprepared according to the invention retained 79-85% of the original drugload even 24 hours after injection into the bloodstream of the animals.The differences between average plasma values of the lipid or drugbetween the liposome and immunoliposome groups were in the range of1.8-13.6% and were close to, or within the range of, assay errors.Probabilities of the null hypothesis between the liposome andimmunoliposome group with regard to drug or lipid values at each timepoint, calculated using Student's t-test, were in the range of0.543-0.938. We conclude that the differences in residual blood levelsof the drug or lipid between the two preparations were negligible andstatistically indistinguishable.

TABLE 21 The amounts of liposome lipid, Topotecan, and of Topotecanremaining encapsulated in the liposomes in the plasma of mice at varioustime points post i.v. injection. Time post Lipid, % of Drug, % ofDrug/Lipid, % of injection injected dose injected dose pre-injectionvalue F5-conjugated liposomal Topotecan (Topo-F5ILs): 1 hour 57.58 ±4.95 55.45 ± 7.23 96.14 ± 7.32 8 hours 35.37 ± 3.84 34.18 ± 5.87  96.31± 11.92 24 hours  15.51 ± 11.84 12.30 ± 9.02 79.36 ± 8.03 LiposomalTopotecan (unconjugated) (Topo-Ls): 1 hour 58.88 ± 9.51 57.63 ± 9.4597.90 ± 5.29 8 hours 39.61 ± 1.99 38.82 ± 1.49 98.06 ± 4.44 24 hours15.84 ± 3.85 13.45 ± 2.64 85.25 ± 7.03

Example 29. Antitumor Efficacy of Liposomal andAnti-HER2-Immunoliposomal Topotecan in BT-474 Xenograft Model

In this study we used the first prototype Topotecan immunoliposomes thatuse triethylammonium-polyphosphate gradient for drug entrapment. Theliposomes were prepared generally following the methods of Examples 11and 19. Lipid matrix components—DSPC (Avanti Polar Lipids; 3 mol.parts), Cholesterol (Calbiochem, 98.3%; 2 mol. parts) andmethoxy-PEG(2000)-DSPE (Avanti Polar Lipids, 0.015 mol. parts)—werecombined with 100% ethanol USP to give the solution containing 0.5 mMphospholipid at 60° C. The ethanol lipid solution was diluted at 60° C.with the aqueous triethylammonium polyphosphate solution (0.608 Mtriethylamine, 0.65 N phosphate, pH 6.1, osmolality 531 mmol/kg), mixedthoroughly, and extruded 10 times through 2 stacked polycarbonatemembranes with the pore size of 100 nm (Nuclepore, Corning) usingthermostatted gas-pressure extruder (Lipex Biomembranes) at 60° C. Theextruded liposomes were chilled on ice, and unencapsulatedtriethylammonium polyphosphate was removed by gel chromatography onSepharose CL-4B using 5% dextrose-5 mM HEPES-Na buffer, pH 6.5, aseluent. The liposome size was 103.8±35.1 nm by QELS. The liposomes inthis buffer were incubated with Topotecan hydrochloride at 60° C. for 30min. at the ratio of 0.35 mg Topotecan base per μmol of phospholipid. Atthe end of incubation, the liposomes were chilled on ice andchromatographed on Sephadex G-75, eluent 20 mM HEPES-Na, 135 mM NaCl, pH6.5, to remove any unencapsulated drug. The drug content was determinedby fluorometry, and the lipid content by phosphate assay as previouslyreported. Liposomal Topotecan so obtained has 365.4±23.1 mg Topotecanbase per mmol of phospholipid. To prepare HER2-targeted Topotecanimmunoliposomes, a portion of this liposomal Topotecan preparation wasincubated with the purified conjugate of anti-HER2 scFv F5 andmaleimido-PEG-DSPE linker generally as described in Example 19. Briefly,F5-PEG-DSPE conjugate in aqueous 10% sucrose-10 mM Na citrate solution,pH 6.5, was combined with Topotecan liposomes at the ratio of 15 mgprotein per mmol of liposome phospholipid, and incubated at 60° C. for30 min. The incubation mixture was chilled on ice and chromatographed onSepharose CL-4B, eluent 20 mM HEPES-Na, 135 mM NaCl, pH 6.5, to removeany unincorporated scFv conjugate. The drug-to-lipid ratio decreased by14% following this additional incubation.

The Topotecan liposome and immunoliposome formulations containing 1-2mg/mL of Topotecan were passed through 0.2 micron sterile syringefilter, dispensed aseptically into polypropylene vials and stored at4-6° C. for up to 1 month before use.

Free Topotecan was freshly prepared by dissolving TopotecanHydrochloride powder at 2 mg/mL in 5% dextrose and sterilized by passagethrough 0.2-micron syringe filter.

A HER2-overexpressing BT-474 human breast adenocarcinoma xenograft modelwas established as described in Example 10. At day 13 post tumorinoculation, the animals having tumors in the range of 120-350 cubic mmwere selected and randomized into 3 treatment and 1 control group of 12animals each. At days 14, 18, and 21 post tumor inoculation the micewere treated with i.v. (tail vein) injections of Topotecan formulationsat the per injection dose of 5 mg/kg body weight, or with equal volumeof physiological saline. General health of the animals was monitoreddaily. Tumor sizes and body weights were monitored twice weekly for upto day 53 post tumor inoculation. The animals whose tumors reached 20%of the body weight, or those with progressive weight loss reaching 20%or more were euthanized.

FIGS. 11 and 12 show the tumor growth and animal body weight data,respectively. Liposomal Topotecan formulations were more active in tumorgrowth suppression than the free drug, and F5-targeted liposomalformulation was more active than the non-targeted one. The average tumorsizes at the end of the observation period were significantly differentamong the treatment groups (p values by non-paired 2-tailed Student'st-test were 1.2×10⁻⁶ for free v. immunoliposomal drug, 0.000114 for freev. liposomal drug, and 0.00718 for liposomal v. immunoliposomal drug).Thus, liposomally encapsulated Topotecan was more active than the freedrug, and anti-HER2 immunoliposomal Topotecan was more active thannon-targeted liposomal drug. In the liposomal and immunoliposomal group,after initial regression, tumor regrowth occurred within 10 days of thelast treatment. There was no tumor regression in the free drug group. Itwas noticed that the liposomal formulations of Topotecan at a given dosewere more toxic than the free drug. There was gastrointestinal toxicity.The animals receiving liposomal Topotecan developed diarrhea andsuffered body weight loss averaging about 14% at its peak. While in thenon-targeted liposomal group the animals recovered, except one (12.5%)that had persistent 15% weight loss at the end of study, in theF5-targeted group five animals (41.6%) developed terminal morbidity andexpired; and two more (16.7%) had persistent weight loss of about 15%.In the control group and free drug group, there was no weight loss ortreatment-related morbidity.

Example 30. Maximum Tolerated Dose (MTD) of Free and Liposomal Topotecanin Mice Given in 3 Weekly i.v. Injections

This study used a liposome Topotecan formulation prepared as in toExample 29, except the triethylammonium polyphosphate solution wasreplaced with triethylammonium sucrose octasulfate solution having 0.65M triethylammonium, pH 6.2; and for extrusion 80-nm polycarbonatemembrane filters were used instead of 100-nm. Volume-weighted liposomesize determined by quasi-elastic light scattering method in Gaussianapproximation (QELS) was 95.1±19.6 nm (average±SD); drug/lipid ratio was369.1±18.3 mg/mmol phospholipid. Five-six week old female Swiss-Webstermice (18-20 g) in the groups of two received three i.v. (tail vein)injections of free or liposomal Topotecan on a once-a-week schedule,starting from the dose of 2 mg/kg Topotecan base per injection andincreasing to each subsequent group by the factor of 1.8 to the dose of37.8 mg/kg. Immunoliposomal Topotecan was not included in this study.Animal body weight and general health was monitored daily. Progressiveweight loss of more than 20% or natural death at any time in any of twoanimals in a group during the period of ten days since the beginning oftreatment were considered indicative of the toxic dose. According to theanimal mortality and weight data MTD was determined to fall within therange of 11.7-21 mg/kg for free Topotecan, and 2.0-3.6 mg/kg forliposomal (Prototype 2) Topotecan. In the second study, the micereceived injections of the free, liposomal, or F5-immunoliposomalTopotecan (prepared from the liposomal Topotecan of this Example asdescribed in Example 29) with the doses from 2.0 mg/kg(liposomal/immunoliposomal Topotecan) or 12 mg/kg (free Topotecan), andincreased to each subsequent group by the factor of 1.15 until the dosenext to the upper range of the established MTD interval was achieved.The highest dose that did not result in death or terminal morbidity inany of the animals was considered an MTD and was found to be 18.4 mg/kgfor free Topotecan, 3.0 mg/kg for liposomal Topotecan, and 3.0 mg/kg forimmunoliposomal Topotecan. Thus, liposomal Topotecan showed greatertoxicity than the free drug.

Example 31. Antitumor Efficacy of Liposomal Topotecan in BT-474Xenograft Model at the Range of 0.125-1.0×MTD

The Topotecan liposomes and F5-immunoliposomes of Example 30 were usedin this study. BT-474 subcutaneous xenografts were raised in nude miceas in Example 29. At day 18 after tumor cell inoculation the animalswith tumors (105-345 cubic mm, average about 200 cubic mm) wererandomized into treatment groups of 6 animals/group, and a control groupof 8 animals/group. The animals received free or liposomal Topotecan at1×MTD, 0.5×MTD, 0.25×MTD, or 0.125×MTD at three i.v. (tail vein)injections at day 19, 23, and 27 post tumor inoculation. The controlgroup received injections of physiological saline. The tumor sizes andanimal body weights were monitored as in Example 29. To obtain animalbody weight measurements, the tumor weight (calculated from the tumorsize assuming tumor density of 1.0) was subtracted from the total animalweight measurements. All drug formulations at MTD showed antitumoractivity (FIGS. 13A-13D). There was no significant difference inefficacy between free and liposomal drug given at their respective MTDor at identical fractions (½, ¼, or ⅛) thereof. Thus, liposomeencapsulation of the drug using TEA-SOS gradient resulted in about6-fold increase in antitumor activity, but also in the similar increasein drug toxicity. Dynamics of animal body weights revealed that alltreatments were non-toxic except the treatment with free Topotecan atMTD which showed transient decrease in body weight (about 15% of thepre-treatment value) that later resolved (FIG. 14).

Example 32. Preparation and Targeted In Vitro Cytotoxicity of TopotecanLiposomes Prepared Using Triethylammonium Sucrooctasulfate EntrapmentMethod

Liposomal Topotecan was prepared generally following the procedure ofExample 18, using the entrapped solution of TEA-SOS having 643 mM TEA,pH 5.7, osmolality 530 mmol/kg, and drug/phospholipid ratio of 170mg/mmol. The liposomes had 155 mg drug/mmol phospholipid; 90% loadingefficiency, and particle size 105 nm. These liposomes were incubatedwith the micellar solution of F5-PEG-DSPE conjugate at about 30 scFv perliposomes (15 mg antibody/mmol phospholipid) at 60° C. for 1 hourgenerally as described in Example 19. Antibody-conjugated liposomes wereseparated by SEC using Sepharose CL-4B and formulated into HBS-6.5HEPES-buffered saline. There was no detectable change in drug/lipidratio during the attachment of anti-HER2 scFv (F5).

The uptake of Topotecan formulations by cancer cells was determined asfollows. HER2-overexpressing human breast adenocarcinoma cells (SK-Br-3,ATCC HTB-30) were plated into 24-well cell culture plates at 150,000cells/well and acclimated overnight. The cells were incubated (intriplicate) with F5-targeted and non-targeted liposomal Topotecan incomplete growth medium at liposome concentrations of 0.1 mM and 0.01 mMfor 4 hours at 37° C. The cells were rinsed 4 times with Hanks' BalancedSalt Solution, solubilized in 0.1% Triton X-100-70% acidifiedisopropanol mixture 1:10, and the amount of cell-associated Topotecanper well was determined by fluorometry. The results (mean±standarderror) are summarized in Table 22. The targeted liposomes delivered100-300 times more drug into the targeted cells than nontargetedliposomes.

TABLE 22 Uptake of liposomal Topotecan by SK-Br-3 breast carcinomacells. Topotecan uptake at Topotecan uptake at 0.1 mM phospholipid, 0.01mM phospholipid, Formulation ng/well ng/well Non-targeted  4.76 ± 0.240.607 ± 0.088 liposome HER2-targeted 533.8 ± 13.7 197.0 ± 4.6  liposomeRatio: Targeted/ 112.1 ± 8.6  324 ± 55  Non-targeted

Cytotoxicity of these Topotecan formulations against SKBr-3 breastcancer cells was determined as described in Example 27. SKBr-3 cellswere inoculated into 96-well plates at 5,000 cells/well, acclimatedovernight, and incubated with increasing concentrations (0.004-30 μg/mL)of free, liposomal, or F5-immunoliposomal Topotecan in cell growthmedium for 4 hours at 37° C. The drug-containing media were removed andthe cells were allowed to grow in the drug-free medium for 72 hours. Thequantity of viable cells per well was determined using Thiazolyl Blue(MTT) tetrazolium assay and expressed as % of that of control(non-treated) cells. The results are presented on FIG. 10. Topotecanimmunoliposomes were more cytotoxic (IC₅₀ 0.15-0.5 μg/mL) thannon-targeted Topotecan liposomes (IC₅₀≥3.1. μg/mL) and free Topotecan(IC₅₀≥2.3 μg/mL)

Example 33. In Vivo Stability of Topotecan Liposomes of Different Size

The liposomes containing TEA-Pn were prepared as in Example 22 usingextrusion 12 times through 100 nm pore size polycarbonate membranes oradditionally 12 times through 50 nm pore size polycarbonate membranes.Topotecan (TPT) was added at a ratio of 150 μg/μmol phospholipid. Theloading was completed at 58° C. for 45 min a hot water bath, followed byquenching on ice. The efficiency of loading for the 50-nm- and100-nm-extruded liposome was 126.80±19.24 μg TPT/μmol PL (84.5±12.8%)and 148.48±10.26 μg TPT/μmol PL (99.0±6.8%), respectively. Female SwissWebster mice in the groups of three were injected intravenously with oneof the two formulations of Ls-TPT at a dose of 5 mg TPT/kg. The micewere sacrificed after 6 h and the blood was collected. Plasma wasanalyzed for TPT and liposome lipid as described in Example 22. Theresults are presented in Table 23.

TABLE 23 In vivo stability of Ls-TPT of different sizes loaded usingTEA-Pn entrapment method. Drug Liposome lipid Drug/lipid Liposome inplasma, % of in plasma, % of ratio, % of pre- size, nm injected doseinjected dose injection value 74.2 ± 21.6 32.93 ± 1.97 45.7 ± 2.2 72.06± 5.51  96.4 ± 29.3 33.26 ± 3.56 37.6 ± 5.3 88.41 ± 15.68

Example 34. Synthesis and Liposome Encapsulation of 6-(3-Aminopropyl)Ellipticine (6-APE)

6-(3-aminopropyl)ellipticine was prepared from ellipticine in a two-stepmethod based on the procedure by Werbel et al., J. Med. Chem. 1986, v.29, p. 1321-1322. 501.4 mg of ellipticine base (NSC 71795) (AldrichChemical Co.) was stirred with approximately 100 mg of sodium hydride(Sigma; washed with anhydrous petroleum ether) in 5 ml of drydimethylformamide (DMF) at room temperature for 30 min. To this mixture,a solution of 678 mg of N-bromopropylphtalimide (Aldrich) in 2 mL of dryDMF was added dropwise. The purple-colored reaction mixture was stirredunder argon overnight, treated with 1 mL of water, and poured into 60 mlof water. The mixture was extracted twice with 25 mL of methylenechloride, the extract was dried over anhydrous sodium sulfate, andpassed through a layer of neutral alumina. The alumina layer was rinsedtwice with 10 mL of methylene chloride and the combined filtrate andrinses were brought to dryness in vacuum. The product was stirredovernight with 20 ml of absolute ethanol and 2 ml of anhydrous hydrazineat room temperature. The obtained slurry was filtered under vacuum, ayellow filtrate was diluted with 50 mL of 0.2 N NaOH and extracted withtwo portions (75 ml and 50 ml) of chloroform. The chloroform extract wasdried over Na₂SO₄ and brought to dryness in vacuum. Crude product (yield408 mg) was chromatographed on silica 60 column eluted isocraticallywith chloroform-methanol mixture (7:3 by volume), saturated with drytrimethylamine. The fractions eluted in a second yellow-colored band,following un-reacted ellipticine, were shown to contain the desiredcompound in approximately 30% yield. The structure was confirmed by¹H-NMR. TLC: R_(f) 0.29-0.31 (Silica 60; CHCl₃-MeOH 7:3 by volume,saturated with trimethylamine). Ellipticine, R_(f) 0.81-0.83. Theobtained compound was converted into dihydrochloride salt by dissolvingin anhydrous ethanol and titration with 6 N HCl solution in dryisopropanol. The orange crystals of 6-APE dihydrochloride (NSC 176328)were filtered out, rinsed with ether, and dried in vacuum. Yield ofdihydrochloride 86%.

The liposomes were prepared by hydration of the neat lipid film of DSPC,Cholesterol, and PEG (M.w. 2,000)-DSPE (3:2:0.015 molar ratio) in asolution of trimethylammonium polyphosphate (TMA-Pn) at 0.5 M TMA, pH5.6, at 60° C., followed by six cycles of rapid freezing (−78° C.) andthawing (60° C.), and extrusion ten times through two stacked 50-nm poresize polycarbonate filters. Unencapsulated TMA-Pn was removed using aSepharose CL-4B column eluted with HEPES-Dextrose (5 mM HEPES, 5%Dextrose, pH 5.5). The liposome size was 85.7±32.1 nm.

Concentrated 6-APE solution (10 mg/ml) was added to theTMA-Pn-containing liposomes at a drug-to-phospholipid ratio of 100 μgAPE/μmol phospholipid, the mixture was incubated at 58° C. for 45 min,and quickly cooled down on ice for 15 min. Unencapsulated drug wasremoved by gel chromatography on a Sephadex G-75 column eluted withHEPES-Dextrose buffer (5 mM HEPES-Na, 5% dextrose, pH 6.5).Liposome-entrapped APE was then quantitated by spectrophotometry as inExample 71, and liposome phospholipid was determined using theextraction assay of Example 70. The drug encapsulation was practicallyquantitative.

Example 35. Preparation of HER2-Targeted Immunoliposomal 6-APE andCytotoxicity of 6-APE Formulations Against HER2-Overexpressing BT-474Breast Cancer Cells In Vitro

Liposomes with encapsulated 6-APE (Ls-APE) were prepared as in Example34 above. Anti-HER2 immunoliposomes with encapsulated 6-APE (F5-ILs-APE)were prepared from Ls-APE by the method of Example 19. An MTT-based cellviability assay of Example 27 was used to determine the cytotoxicity of6-APE delivered as a solution, Ls-APE, or as HER2-targeted F5-ILs-APEagainst HER2-overexpressing human breast carcinoma cells (BT-474). Thecells were exposed to drug-containing media for 6 hours, andpost-incubated in drug-free medium for 3 days. The results are shown onFIG. 15. The IC₅₀ for free APE is 0.26 APE/ml, for F5-ILs-APE was 0.756μg APE/ml, and for nontargeted Ls-APE was 51.0 APE/ml. There was a 67.5fold difference in activity between targeted and nontargeted liposomal6-APE, indicating a considerable targeted delivery effect.

Example 36. EGFR-Targeted Immunoliposomal Formulations of 6-APE andCytotoxicity Against Cancer Cells In Vitro

6-APE-loaded liposomes were prepared as described in Example 34.EGFR-targeted immunoliposomes were prepared by attachment ofEGFR-specific Fab′ antibody fragments as follows. An EGFR-specific IgGMAb C225 (cetuximab, ERBITUX™, Imclone Systems) was digested with pepsinto produce (Fab′)₂ fragments. Purified (Fab′)₂ fragments were reduced bytreatment with 10-20 mM 2-mercaptoethylamine for 15 min at 37° C., andFab′ fragments were purified by gel filtration using Sephadex G-25. Thepresence of reactive thiol groups was typically about 0.9 thiol groupsper protein molecule (quantified using Ellmann's reagent). C225Fab′ werecovalently conjugated to an amphiphilic linker Mal-PEG-DSPE (AvantiPolar Lipids, AL) in aqueous solution at pH 6.2-6.5 and protein-linkermolar ratio of 1:4 for 2-4 hours at room temperature, or overnight at4-6° C., to produce C225Fab′-PEG-DSPE conjugate with the yield 30-50% ofthe protein. This micelle-forming conjugate was separated fromnon-reacted protein by size exclusion column chromatography on 3%agarose—4% polyacrylamide beaded gel (Ultrogel AcA34, obtained fromSigma Chemical Co.), eluted with HBS-6.5 buffer. The conjugate wasrecovered in void volume fractions. Immunoliposomal 6-APE was formed byincubating these liposomes with C225 Fab′-PEG-DSPE with drug-loadedliposomes at the ratio of 30 mg C225 protein/mmol liposome phospholipidfor 30 min at 60° C., quenching on ice for 15 min, and purifying theimmunoliposomes by gel chromatography on a Sepharose CL-4B column alsoeluted with HBS-6.5 buffer (the liposomes appear in or near the voidvolume of the column).

MDA-MB-468 EGFR-overexpressing human breast cancer cells and MCF-7 humanbreast cancer cells with low EGFR expression (ATCC, Rockville, Md.) werecultured in their supplier-recommended growth media, and thecytotoxicity of free, liposomal, and anti-EGFR-immunoliposomal 6-APEagainst these cells was studied according to the method of Example 27The cells were incubated with drug-containing media for 6 hours,followed by 3 days post-incubation in the drug-free medium. The resultsare shown in FIG. 16. In MDA-MB-468 cells IC₅₀ for the free 6-APE wasabout 0.1 μg/ml, and for C225-ILs-APE about 0.9 μg/ml. In MCF-7 cellsIC₅₀ was about 0.1 for the free 6-APE was about 0.5 μg/ml, and forC225-ILs-APE about 14 μg/ml. IC₅₀ of Ls-APE in both cell lines was >30μg/ml. Thus, EGFR-targeted 6-APE-loaded immunoliposomes demonstratedantigen-specific cytotoxic activity in EGFR-overexpressing MDA-MB-468breast cancer cells, but not in MCF-7 breast cancer cell that do notoverexpress EGFR. In MCF-7 cells, the targeted and nontargeted 6-APEliposomes were equally active.

Example 37. Pharmacokinetics of Liposomal 6-APE in Rats

Liposomes with entrapped TEA-Pn solution (557 mM phosphate groups, 500mM TEA, pH 5.8, osmolality 480 mmol/kg) and lipid composition of DSPC,cholesterol, and PEG-DSPE (molar ratio 3:2:0.015) were prepared as inExample 11 above. Ethanolic solution of the lipids was combined at 60°C. with 10 volumes of the aqueous TEA-Pn solution, and extruded tentimes through two stacked 80 nm pore size polycarbonate membranes.Unencapsulated TEA-Pn was removed using a Sepharose CL-4B column elutedwith IVIES-Dextrose (5 mM IVIES-Na, 5% Dextrose, pH 5.5). The liposomesize was 92.3±23.3 nm by QELS. A non-exchangeable radioactive lipidlabel [³H]-CHE was included in the lipid matrix at 0.5 mCi/mmolphospholipid. The liposomes were loaded with 6-APE as described inExample 34.

The pharmacokinetic study followed the protocol of Example 9. Female SimAlbino rats (9 weeks, 200 g) were injected i.v. at a dose of 10 mg6-APE/kg. Blood was drawn at prescribed time points and the plasma wasanalyzed for 6-APE by fluorometry. Plasma aliquots (0.05-0.2 ml) weremixed with 1-2 mL of 90% aqueous isopropanol-0.1 N HCl, and the 6-APEwas quantified by fluorescence as in Example 71. The lipid wasquantified by [³H]-CHE radioactivity scintillation counting.

The results are shown in FIG. 17. The blood half-life (t_(1/2)) of thedrug was 13.7 hours and of the liposome lipid 16.6 hours (panel A). Thehalf-life of the drug release from liposomes was 77.9 hours,demonstrating remarkable encapsulation stability (panel B).

Example 38. Synthesis and Liposomal Encapsulation of2-(2-(N,N-diethylamino)ethyl)ellipticinium (2-DAE)

2-(2-(N,N-diethylamino)ethyl-ellipticinium chloride (NSC 359449) is ananti-cancer ellipticine derivative which is prepared by alkylation ofellipticine with 2-(N,N-diethylamino)ethylchloride in methanol in thepresence of triethylamine (see Werbel, L. M., Angelo, M., Fry, D. M.,and Worth, D. F. J. Med. Chem. 1986, 29:1321-1322). Liposomes containingentrapped TEA-Pn were prepared as described in Example 37. 2-DAE.2HClwas incubated with the TEA-Pn liposomes in 5 mM HEPES-Na, 5% Dextrose,pH 7.4, at a 2-DAE-to-phosholipid ratio of 100 μg/μmol. The amount ofloaded drug was 88.2 μg APE/μmol PL (efficiency 88.2%).

Example 39. Pharmacokinetics of Liposomal 2-DAE in Rats

Blood pharmacokinetics of liposomal 2-DAE (Example 38) was studied inrats as in Example 37. The t_(1/2) of 2-DAE was 17.8 h and of theliposome lipid matrix, 18.2 h (A). The half-life of the drug releasefrom liposomes in the blood was t_(1/2)=677 h (B). Thus, these liposomeswere extraordinarily stable against drug leakage in the bloodstream.

Example 40. Loading of Vinorelbine into Liposomes Using TEA-Pn Method.The Effect of pH

The liposomes were prepared by the ethanol injection method as inExample 11 using TEA-Pn solution of 0.608 M TEA, 0.65 M phosphategroups, pH 6.1, and osmolality 531 mmol/kg, and lipid suspensionextrusion 15 times through two stacked 100 nm pore size polycarbonatemembranes. The resulting liposome size was 108.3±17.1 nm by QELSVinorelbine (VRB) in the form of stock solution of vinorelbinebitartrate 10 mg/mL USP was added to the liposomes in aqueous 5 mMHEPES-Na, 5% dextrose, pH 6.5, at a drug-to-phospholipid ratio of 350μg/μmol, the pH was adjusted to the desired value using 1-5 N NaOH. andthe mixture was incubated at 58±2° C. for 30 min. The mixture was thenchilled on ice for 15 min, and unencapsulated drug was removed bySephadex G-75 gel filtration chromatography, eluting with HBS-6.5 buffer(20 mM HEPES-Na, 135 mM NaCl, pH 6.5). Aliquots of purified liposomeswere then solubilized in acid isopropanol and analyzed for vinorelbineusing spectrophotometry at 270 nm. Liposome phospholipid was quantifiedusing the phosphate assay of Bartlett (1959) after methanol-chloroformextraction.

The calculated drug-to-lipid ratios after loading were as shown in Table24. Vinorelbine loading was quantitative (i.e. practically 100%) andindependent of pH in the studied range.

TABLE 24 Vinorelbine loading into liposomes with entrapped TEA-Pn atvarious pH values of external buffer Drug-to-phospholipid Loading pHratio (μg/μmol) efficiency (%) 4.5  351.2 ± 52.88  100.4 ± 15.2 5.0347.6 ± 6.35  99.3 ± 1.8 5.75 355.2 ± 11.2 101.5 ± 3.2 6.25 377.0 ± 21.5107.7 ± 6.6 7.0  374.3 ± 29.58 106.9 ± 9.0

Example 41 Liposomal Vinorelbine Prepared by TEA-Pn Method at VariousDrug/Lipid Ratios: Encapsulation Efficiency and In Vivo Stability inMice

Liposomes with entrapped TEA-Pn solution were prepared according toExample 40 except that [³H]-CHE was included in the lipid matrix at 1.5mCi/mmol phospholipid. The liposome size was 98.5±34.3 nm by QELS. Theliposomes were mixed with vinorelbine bitartrate USP in aqueous bufferof 5 mM HEPES-Na, 5% dextrose, pH 6.5 at the drug-to-phospholipid ratioof 150-450 mg VRB/mmol, and incubated at 58±2° C. for 30 min. No pHadjustment was made following the addition of the drug. Thevinorelbine-loaded liposomes (Ls-VRB) were isolated and analyzed for thedrug and phospholipid as in Example 40.

Female five-six week old Swiss Webster mice (Harlan Bioresearch) in thegroups of three were injected intravenously with Ls-VRB-Pn at a dose of5 mg VRB/kg. The lipid dose varied according to the degree of loadingand can be determined from the above calculated drug-to-lipid ratios. At8 hours or 24 hours post injection, the animals were anesthetized,exsanguinated, and the blood was collected on ice into weighed tubescontaining known amounts of PBS with 0.04% EDTA. The blood cells wereseparated by centrifugation, and the supernatants were analyzed forliposome lipid by [³H]-CHE radioactivity scintillation counting and forvinorelbine using HPLC as follows. The samples were spiked withvinblastine (internal standard), extracted with diethyl ether,evaporated, and the residues were dissolved in the mobile phaseconsisting of aqueous 50 mM triethylammonium acetate (pH 5.5) andacetonitrile (58:42 by volume) The samples were loaded on a C18 reversephase silica column (Supelco C-18 column, 250 mm×4 mm i.d., particlesize of 5 μm) preceded by a C-18 guard column. The column was elutedisocratically with the above mobile phase at a flow rate of 1.0 ml/min.VRB was detected using an absorbance detector at 280 nm. Typicalretention times for VRB and vinblastine (internal standard) were 9.1 minand 7.8 min, respectively.

The results are shown in Table 25. The loading efficiency decreased withthe increase in drug/lipid ratio, from practically 100% at 150 mg/mmolto about 66% at 450 mg/mmol. It was noted that the addition ofvinorelbine bitartrate at the ratios of over 250 mg vinorelbine per mmolphospholipid caused substantial acidification of the liposome suspension(pH<4.0) that lead to reduced loading efficiency. Thus, the need for pHcontrol during the drug loading step was established. The amounts ofliposome matrix detected in the blood after 8 hours were in the range of30.4±6.6% of injected dose (% id) to 38.6±5.2% id without apparentrelation to the absolute amount of injected lipid. After 24 hours therewas still from 6.4% ID to 14.8% ID of the lipid matrix detectable in theblood. The amount of drug that remained encapsulated after 8 hoursvaried from 37% to 63%. However, as 24 hours post injection the druglevels dropped below detection limit of the employed analytical method.

TABLE 25 Encapsulation efficiency and in vivo drug retention ofliposomal vinorelbine prepared at different drug/lipid ratios usingTEA-Pn method (without loading buffer pH adjustment). The drug retentiondata are mean ± SD (N = 3). Vinorelbine/phospholipid ratio Input,Output, Encapsulation % drug remaining mg/mmol, mg/mmol, efficiency,encapsulated at 8 calculated measured % hours post injection 150 156104.0 36.6 ± 4.2 250 238 95.2 53.3 ± 1.3 350 260 74.3 65.9 ± 2.3 450 29966.4 63.0 ± 4.1

Example 42. Vinorelbine Loading into Liposomes Using TEA-SOS Method atVarious Drug/Lipid Ratios

TEA-SOS liposomes for drug loading were prepared as in Example 40 exceptthat the TEA-SOS solution with 0.65 M TEA, pH 5.4, osmolality 521mmol/kg was used instead of TEA-Pn solution, and the liposomes wereextruded through 80 nm pore size polycarbonate membranes. The liposomesize was 86.6±12.9 nm by QELS. VRB was added to the liposomes in aqueous5 mM HEPES-Na, 5% Dextrose, pH 6.5, at various drug-to-phospholipidratios, and the mixture was subsequently incubated at 60° C. for 30 min.The VRB-loaded liposomes were then isolated and analyzed as in Example40.

The calculated drug-to-lipid ratios in the VRB liposomes are shown inTable 26. Remarkably, as opposed to polymeric anion assisted loading,vinorelbine loading in the liposomes with polyanionized sugar (sucroseoctasulfate) was practically quantitative independently of thedrug/lipid ratio for up to 450 mg VRB/mmol phospholipid, and onlyslightly less (88%) at 550 mg VRB/mmol phospholipid.

TABLE 26 Dependence of vinorelbine loading into liposomes ondrug-to-lipid ratio Vinorelbine/phospholipid ratio, mg/mmol EncapsulatedLoading Total into liposomes efficiency (%) 150 159.9 ± 11.5 106.6 ± 8.1250  255. ± 12.4 102.2 ± 5.1 350 381.8 ± 16.3 109.1 ± 5.1 450 456.1 ±29.5 101.4 ± 6.6 550 486.2 ± 26.0  88.4 ± 4.2

Example 43. Preparation of HER2-Targeted Immunoliposomes Loaded withVinorelbine by TEA-Pn Method, and Comparative Blood Pharmacokinetics ofHER2-Targeted and Nontargeted Vinorelbine Liposomes in Rats

Anti-HER2 scFv F5-PEG-DSPE conjugate was prepared as in Example 19.HER2-targeted vinorelbine immunoliposomes were prepared by incubation ofnon-targeted vinorelbine liposomes (Example 41, loaded atdrug/phospholipid ratio of 350 mg/mmol) with F5-PEG-DSPE conjugate(Example 19) in aqueous 20 mM HEPES-Na, 135 mM NaCl, pH 6.5 buffer atthe protein/phospholipid ratio of 15 mg/mmol at 60° C. for 30 min.Unincorporated F5 conjugate was removed by gel chromatography on aSepharose 4B column eluted with the same buffer. Non-targeted liposomes(Ls-Pn-VRB) and HER2-targeted ones (F5-ILs-Pn-VRB) were administeredi.v. to female Albino rats (8-9 weeks old; 200 g) at a dose of 5 mgVRB/kg. At various time points, blood was collected as described inExample 9, and analyzed for VRB and the liposome lipid as in Example 41.Blood half-life of the liposome lipids and the 50% drug release timewere calculated from the lipid concentration-time plots or by drug/lipidratio-time plots, respectively, by finding best fit to monoexponentialkinetics using the MICROSOFT EXCEL (Microsoft Corp.) spreadsheet TRENDfunction. The results (FIG. 18) indicated that both targeted andnon-targeted vinorelbine liposomes had identical drug and lipidpharmacokinetics with lipid half-life of about 12.1 hours and 50% drugrelease time of about 4.3 hours.

Example 44. Preparation and Comparative In Vivo Stability of VinorelbineLiposomes Prepared Using Ammonium and Substituted Ammonium Salts

Ammonium dextran sulfate (DS-A) solution with pH 5.8, 0.65 M NH₄ ⁺,osmolality of 390 mmol/kg, and triethylammonium dextran sulfate solution(DS-TEA) with pH 6.0, 0.65 M NH₄ ⁺, osmolality 465 mmol/kg, wereprepared from Dextran sulfate with mol. weight 10,000 (Sigma ChemicalCo.) according to the method of Example 4, using titration with 12.4 Maqueous ammonia or neat triethylamine, respectively. Ammonium sulfate(S-A) aqueous solution 325 mM, pH 5.1, osmolality 703 mmol/kg, wasprepared from analytical grade ammonium sulfate. All three solutionscontained less than 1% Na⁺ of the total cation content. Liposomesentrapping these solutions were prepared using the ethanolmixing-extrusion method of Example 41 (DSPC/Cholesterol/PEG-DSPE3:2:0.015 molar ratio). Radioactive lipid label [³H]-CHE was included inthe lipid matrix at 1.5 mCi/mmol phospholipid. Extrusion step consistedof 10 passages through two stacked 0.1 μm polycarbonate membranes. VRBwas added to the liposomes in 5 mM HEPES-Na, 5% Dextrose, pH 6.5, at adrug-to-phospholipid ratio of 350 mg/mmol, the pH was adjusted to 6.5using 1 N NaOH, and the mixture was incubated at 58-60 C ° C. for 30min. The reaction was then chilled on ice for 15 min, and unencapsulateddrug was removed using Sephadex G-75 gel filtration chromatography,eluting with aqueous 20 mM HEPES-Na, 135 mM NaCl, pH 6.5. The purified,vinorelbine-loaded liposomes were analyzed for VRBspectrophotometrically and for phospholipid using the phosphate assay ofBartlett (1959) (see Examples 70, 71). Blood pharmacokinetics of theliposomal lipid and drug was studied in rats as in Example 43.

The results are shown in FIGS. 19-20, and in Table 27. Liposomes loadedwith triethylammonium dextransulfate were compared with those loadedusing ammonium salt of dextran sulfate. Unexpectedly, those loaded usingthe triethylammonium salt were considerably more stable than thoseloaded using ammonium salt. The pharmacokinetics of the liposomalcarrier itself was similar with the three different formulations and wasthus primarily dependent on the lipid composition employed. Leakage ofvinorelbine from Ls-VRB loaded using triethylammonium dextran sulfatewas about three times slower than from those loaded using ammoniumdextransulfate. The liposomes loaded using ammonium sulfate had thefastest drug leakage rate.

TABLE 27 Comparative in vivo stability of drug encapsulation intoliposomes using entrapped ammonium and substituted ammonium salt.Formulation, Liposome size, Blood half-life Time to 50% liposome- nm,mean ± SD of the lipid drug release in entrapped salt (by QELS) matrix,hours the blood, hours DS-TEA 120.8 ± 28.5  9.5 ± 3.3 66.3 ± 13.4 DS-A107.8 ± 15.4 11.2 ± 0.6 22.9 ± 1.7  S-A 114.5. ± 15.6  10.7 ± 0.2 1.77 ±0.16

Example 45. Preparation and In Vivo Stability of Vinorelbine LoadedLiposomes of Various Size

[³H]-CHE-labeled liposomes (1.5 mCi/mmol phospholipid) with entrappedsolution of triethylammonium sucrose octasulfate (0.65 M TEA, pH 6.4,osmolality 502 mmol/kg) were prepared by the ethanol mixing-extrusionmethod of Example 11. The extrusion step contained 15 passages throughtwo stacked polycarbonate membranes with the pore size of 0.05, 0.08, or0.1 μm. Vinorelbine loading, isolation of vinorelbine liposomes, andliposome characterization followed the method of Example 40. FemaleAlbino rats (8-9 weeks old; 200 g) were used to study liposome in vivostability. Liposome lipid and drug pharmacokinetics was studied in ratsas in Example 43.

The results are shown in FIGS. 21, 22, and in the Table 28 below.Liposomes extruded through 0.05, 0.08, and 0.1 μm polycarbonate filterswere compared and shown to have similar drug and liposomal carrierpharmacokinetics, as well as a similar extent of contents leakage. Thedrug release from the liposomes in blood was characterized by the 50%release times in the range of approximately 40-80 hours, well above 24hours.

TABLE 28 Characterization of vinorelbine liposomes. Liposome Blood half-Time to 50% size, nm, Drug load, Loading life of the drug release mean ±SD mg/mmol efficiency, lipid matrix, in the (by QELS) phospholipid %hours blood, hours 87.6 ± 28.1 352.4 ± 13.9 100.7 ± 4.0 14.6 ± 0.7 39.7± 3.1 98.5 ± 15.1 322.6 ± 22.7  92.2 ± 6.5 13.0 ± 0.2 47.9 ± 3.8 109.6 ±24.6  357.0 ± 10.5 102.0 ± 3.0 14.3 ± 0.3 78.0 ± 1.4

Example 46. Preparation of HER2-Targeted Vinorelbine Liposomes UsingTEA-SOS Entrapment Method, and Pharmacokinetics of HER2 scFv-Targetedand Non-Targeted Immunoliposomal Vinorelbine in Rats

Liposomes were prepared, loaded with vinorelbine at the 350 mg/mmoldrug-phospholipid ratio, and analyzed as described in Example 43, exceptthat TEA-SOS solution of Example 45 was substituted for TEA-Pn solution.Extrusion step included 15 passages through 0.08 μm pore sizepolycarbonate filters. The liposome size was 95.0±26.0 nm by QELS.F5scFv-linked anti-HER2 vinorelbine immunoliposomes were prepared fromthese vinorelbine liposomes, and blood pharmacokinetics of the liposomallipid and drug of HER2-targeted and nontargeted liposome vinorelbine wasstudied in rats as described in Example 43. Circulation half-life of theliposome lipid was 11.4 hours and 10.3 hours, and the 50% drug releasetime was 30.9 hours and 30.3 hours for F5-ILs-VRB and Ls-VRB,respectively. Thus, the lipid and drug pharmacokinetics of Ls-VRB andF5-ILs-VRB was very close, indicating that the introduction of thescFv-PEG-DSPE conjugate neither affected clearance of the carrier itselfnor resulted in increased leakage of the drug from the carrier while inthe circulation (FIGS. 23, 24).

Example 47. Preparation and Pharmacokinetic Properties of VinorelbineLiposomes Comprising Non-Ionic Lipid Derivatives of Poly(EthyleneGlycol)

Methoxy-PEG (Mol. weight 2,000)-derivative of synthetic C₂₀-ceramide(PEG-ceramide) was obtained from Northern Lipids, Inc., Canada.Methoxy-PEG (Mol. weight 2,000)-distearoylglycerol (PEG-DSG) (SUNBRIGHTGS20) was from NOF Corp., Japan.

Liposomes having the lipid composition of DSPC, cholesterol, andPEG-lipid (PEG-ceramide or PEG-DSG) in the molar ratio of 3:2:0.3 andentrapped TEA-SOS solution (0.65 M TEA, pH 6.4, osmolality 502 mmol/kg)were prepared by the ethanol mixing/extrusion method of Example 11. Theextrusion step included two passages through two stacked polycarbonatemembrane filters 2 times using pore size 0.2 μm and 10 times using 0.08μm pore size. The liposomes were loaded with vinorelbine at thedrug/phospholipid ratio of 350 mg/mmol, characterized by size, drug, andlipid concentration, and their pharmacokinetics was studied in rats asin Example 46. Both formulations showed prolonged circulation time ofthe lipid matrix and slow release of the drug in vivo, with at least 50%of the drug remaining encapsulated after 24 hour in the blood in vivo,as shown in the Table 29 below.

TABLE 29 Characterization of vinorelbine liposomes with variousPEG-lipids. Liposome size, Drug load, Loading Blood half-life Time to50% nm, mean ± SD mg/mmol efficiency of the lipid drug release inPEG-lipid (by QELS) phospholipid % matrix, hours the blood, hoursPEG-ceramide 103.3 ± 30.9 291.4 ± 18.0 83.26 ± 5.14 14.0 102.7 PEG-DSG101.3 ± 20.1 359.3 ± 7.2  102.7 ± 2.1  15.1 24.6

Remarkably, the increased PEGylation of these liposomes (PEG lipidcontent about 5.7 mol. % of the total lipid) had practically no effecton the liposome blood circulation longevity compared to the similar,size-matched liposomes having low PEGylation of about 0.3 mol. % oftotal lipid (Example 45, 109.6 nm, t_(1/2)=14.3 hours; 98.5 nm,t_(1/2)=13.0 hours).

Example 48. Preparation of HER2-Targeted Liposomal Vinorelbine andCytotoxicity of Free, HER2-Targeted, and Non-Targeted LiposomalVinorelbine Against MDA-MB-453 Cells In Vitro

Vinorelbine-loaded liposomes (Ls-VRB) were prepared as in Example 42(without [³H]-CHE) using drug loading at pH 6.0 and 350 μgvinorelbine/μmol phospholipid. Anti-HER2 immunoliposomal vinorelbine(F5-ILs-VRB) was formed by incubating these liposomes with F5-PEG-DSPEconjugate as described in Example 19 and 42 above, except that [³H]-CHEwas not added. “Free” vinorelbine was prepared by dilution ofvinorelbine bitartrate 10 mg/ml solution USP into the cell culturemedium.

MDA-MB-453 are human breast adenocarcinoma cells (American Type CultureCollection, Rockville, Md.) that moderately overexpresses HER2 receptor(about 3×10⁴ to 1×10⁵ copies/cell). Cytotoxicity of VRB delivered as thefree drug, as nontargeted liposomal vinorelbine, or as HER2-targeted(F5)-immunoliposomal vinorelbine against MDA-MB-453 cells was determinedas described in Example 27, except that the cells were plated in 96 wellmicrotiter plates under the supplier-recommended growth conditions(Leibowitz L-15 with 10% fetal calf serum, no CO₂ supplementation) at adensity of 10,000 cells/well, and the drug formulations were added in aseries of 1:3 stepwise dilutions starting with 0.03-0.1 mg/ml. The cellviability data were plotted against drug concentration (FIG. 25) anddrug concentrations required to reduce the cell viability to 50% (IC₅₀)were estimated from the graphs. IC₅₀ of F5-targeted vinorelbine liposome0.06 μg/ml) was close to that of the free drug (0.07 μg/ml) andsubstantially lower than that of non-targeted liposomes (2.2 μg/ml).This represents a 37-fold enhancement in activity as a result of cancercell-specific targeted delivery of the drug.

Example 49. Cytotoxicity of Free, HER2-Targeted, and Non-TargetedLiposomal Vinorelbine Against CaLu-3 Cells In Vitro

The liposomes and methods of the previous example (Example 48) were usedto study cytotoxicity of free vinorelbine, Ls-VRB, and F5-ILs-VRB inHER2-overexpressing human non-small cell lung carcinoma cells CaLu-3(American Type Culture Collection, Rockville, Md.). The cells were grownin RPMI-1460 medium with 10% fetal calf serum in the presence of 5% CO₂.The results are shown in FIG. 26. The IC₅₀ for free VRB was 1.2 μg/ml,10 μg/ml for F5-ILs-VRB, and 50 μg/ml for nontargeted Ls-VRB. Thisrepresents a 5-fold enhancement in liposome-encapsulated drug activityas a function of targeted delivery to the cells.

Example 50. Cytotoxicity of Free, HER2-Targeted, and Non-TargetedLiposomal Vinorelbine Against SKBr-3 Cells In Vitro

The liposomes and methods of Example 48 and the were used to studycytotoxicity of free vinorelbine, Ls-VRB, and F5-ILs-VRB inHER2-overexpressing human breast carcinoma cells SKBr-3 (American TypeCulture Collection, Rockville, Md.), except that the cells were grown inthe modified McCoy 5A medium with 10% fetal calf serum in the presenceof 5% CO₂, plated at a density of 5,000 cells/well, and the drug wasincubated with the cells for 6 h.

The results are shown in FIG. 27. The IC₅₀ for free VRB was 0.28 μg/ml,0.17 μg/ml for F5-ILs-VRB, and 0.8 μg/ml for nontargeted Ls-VRB. Thisrepresents a 4.7-fold enhancement in drug activity as a function oftargeted delivery.

Example 51 In Vivo Antitumor Efficacy of Liposomal Vinorelbine in HT29Human Colon Cancer Xenografts in Mice

Small unilamellar vesicle liposomes (93.2±26.4 nm by QELS) were preparedfrom distearoylphosphatidylcholine, cholesterol, and PEG-DSPE (3:2:0.045molar ratio) by hydration from a concentrated ethanolic solution in anaqueous solution of triethylammonium sucroseoctasulfate (0.6 Mtriethylammonium, pH 5.7-6.2), followed by repeated extrusion throughpolycarbonate membranes (100 nm pore size), removal of extraliposomalpolyanionic salt, and loading with vinorelbine by incubation with theliposomes in isoosmotic buffer pH 6.5, drug/lipid ratio of 325 mgVRB/mmol phospholipid, at 60° C. as described in Example 42.

Female BALB/c homozygous nude mice (6-8 weeks, weighing 17-20 g) wereinjected subcutaneously in the flank area with 1×10⁶ of HT-29 humancolon carcinoma cells (American Type Culture Collection, Rockville,Md.). Starting with day 16 post-tumor inoculation, when the mean tumordiameter reached 5-8 mm, the mice were randomly divided into threegroups of six animals each and treated with free or liposomalvinorelbine at a dose of 5 mg/kg through the tail-vein every three daysfor a total of four injections. For the control group, mice were treatedwith an equal volume of saline. The tumor size of each mice was measuredusing a caliper and the tumor volume was calculated using the formula:(tumor length)×(tumor width)²/2. To assess treatment-related toxicity,the animals were also weighed twice weekly. Liposomal vinorelbine wasshown to be considerably more efficacious in suppressing the growth ofHT-29 tumors that free vinorelbine, causing tumors to regress, while inthe free drug group the tumors always continued to grow (FIG. 28). Therewas little change in animals' body weight during the course of treatmentindicating that the treatment was well tolerated, and thatliposomalization did not increase the drug toxicity (FIG. 29).

Example 52 In Vivo Antitumor Efficacy of Liposomal Vinorelbine AgainstC-26 Syngeneic Murine Colon Cancer Tumors

Liposomal vinorelbine and free vinorelbine were prepared as in Example48. Male BALB/c mice (6-8 weeks, weighing 17-20 g) were inoculatedsubcutaneously with 2×10⁵ of C-26 murine colon carcinoma cells. At day17 post-inoculation, when the mean tumor diameter reached 5-8 mm, micewere randomly divided into six treatment groups of five animals/group.The tumor bearing mice were injected through the tail-vein with freevinorelbine at 6 mg/kg, 8 mg/kg, or 12 mg/kg, and with liposomalvinorelbine at 4 mg/kg or 6 mg/kg every three days for a total of fourinjections. For the control group, mice were injected with equal volumeof normal saline. Tumor sizes and animals body weights were followed asin Example 51. Liposomal vinorelbine even at 4 mg/kg, was considerablymore efficacious in reducing the tumor growth than free drug at 12 mg/kg(FIG. 30), The animal body weights in the course of treatment showedlittle change (<10% decrease) indicating that the toxicity of liposomalvinorelbine was not increased compared to that of free drug (FIG. 31).

Example 53 In Vivo Antitumor Efficacy of HER2-Targeted LiposomalVinorelbine Against BT-474 Human Breast Cancer Xenograft Tumors in Mice:Effect of Loading Counter-Ion

VRB-loaded liposomes 99.5±10.2 nm in size were prepared by the TEA-Pnmethod of Example 41 and TEA-SOS method of Example 42, respectively,except that [³H]-CHE was not added. VRB was loaded at thedrug/phospholipid ratio of 350 mg/mmol. HER2-targeted liposomalvinorelbine was formed by incubating these liposomes with F5-PEG-DSPEconjugate (see Example 19) as described in Example 43. BT-474HER2-overexpressing human breast carcinoma xenografts were raised inhomozygous nude mice as in Example 10. At day 25 post tumor cellinoculation, when the tumors reached about 200 mm³ in size (range144-309 mm³), the mice were randomized into four groups of eightanimals/group, and treated i.v. with 5 mg/kg of free VRB, F5-ILs-VRBwith Pn as a counter-ion, or F5-ILs-VRB with SOS as a counter-ion, at adose of 5 mg/kg weekly for a total of three injections. The controlgroup received equal volume of normal saline. The tumors and animal bodyweights were monitored as in Example 10. HER2-targeted liposomalvinorelbine loaded using sucrose octasulfate was noticeably moreefficacious in reducing tumor growth than the same targeted constructloaded using poly(phosphate), and both immunoliposomal preparations wereconsiderably more efficacious than free vinorelbine when administered ata dose of 5 mg VRB/kg (FIG. 32). The drug-treated mice demonstratedlittle change in weight indicating that the treatment was well tolerated(FIG. 33).

Example 54. In Vivo Antitumor Efficacy of HER2-Targeted LiposomalVinorelbine Against BT-474 Human Breast Cancer Xenograft Tumors in Mice:Effect of PEGylation

The liposomes of DSPC and cholesterol in the molar ratio 3:2 wereprepared according to Example 48 by hydration of the lipid matrix ofDSPC, cholesterol, and PEG-distearoylglycerol with PEG mol. weight 2,000(GS-20, NOF Corp., Japan) at a molar ratio 3:2:0.015 (“0.5% PEG”) or3:2:0.3 (“10% PEG”) via ethanolic solution method in an aqueoustriethylammonium sucroseoctasulfate, followed by membrane extrusionaccording to Example 48. VRB was loaded into the liposomes at thedrug/phospholipid ratio of 350 mg/mmol. F5 immunoliposomal vinorelbinewas formed by incubating these liposomes with F5-PEG-DSPE conjugate(Example 19) as described in Example 43. Nude mice with BT-474xenografts were raised and treated i.v. with free VRB, F5-ILs-VRB-“0.5%PEG”, or F5-ILs-VRB-“10% PEG” at 5 mg/kg as in Example 53. As shown inFIG. 34, F5-ILs-VRB with higher PEGylation provided with a non-ionic PEGlipid derivative PEG-DSG was noticeably more efficacious in reducingtumor growth than F5-ILs-VRB with lower amount of PEG-DSG, while bothpreparations were more active than the free drug.

Example 55 In Vivo Antitumor Efficacy of EGFR-Targeted LiposomalVinorelbine Against U87 Human Brain Cancer Xenograft Tumors in Mice

The liposomes (86.6±12.9 nm in size by QELS) with encapsulated 0.65 MTEA-SOS solution were prepared and loaded with VRB according to Example42. Anti-EGFR-immunoliposomal VRB (C225Fab′-ILs-VRB) was prepared byincubation of VRB liposomes with the PEG-DSPE conjugate of an anti-EGFRantibody Fab′ fragments as described in Example 36.

Male NCR nu/nu mice (5-6 weeks, weighing 17-20 g) were injectedsubcutaneously in the flank area with 1×10⁷ of U87 human glioblastomacells (ATCC) suspended in the growth medium in a total volume of 150 μl.When the tumor reached an average size of 250 mm³, mice were randomlydivided into four groups of 10-12 animals. The mice were treated withthree weekly i.v. injections of “free” VRB, nontargeted Ls-VRB, orC225Fab′-ILs-VRB at a dose of 5 mg VRB/kg. The control group received anequal volume of saline. The tumor sizes and animal body weights weremonitored as in Example 10. C225-Fab′-ILs-VRB was noticeably moreefficacious in suppressing the growth of EGFR-overexpressing human braincancer xenograft tumors than either non-targeted liposomal vinorelbineor free vinorelbine at an equal dose (FIG. 35).

Example 56. Preparation and Pharmacokinetics of Doxorubicin Encapsulatedin the Liposomes Using Triethylammonium Sulfate Method

Liposomes with various lipid matrix composition (as indicated in thetable below) were formed as described in Example 2. N-Glutaryl-DSPE(Glu-DSPE) was from Avanti Polar Lipids, AL, USA. A neat lipid film wasformed from the lipid solution in chloroform using rotary evaporation,trace volatiles were removed under vacuum (90 μm Hg, 2 hours), the lipidfilm was hydrated in a triethylammonium sulfate (TEA-504) solution (0.65N TEA), subjected to six cycles of rapid freeze and thaw, and extrudedthrough two stacked 0.1 μm pore size polycarbonate filters ten times andthrough two stacked 0.05 μm pore size polycarbonate filters ten times.For lipid matrix quantification in the blood samples, [³H]-CHE wasincluded in the lipid matrix at 0.5-1.5 mCi/mmol phospholipid. Theliposomes with entrapped TEA-504 solution were loaded with doxorubicinaccording to Example 2. The liposomes in HEPES-buffered saline (20 mMHEPES-Na, 135 mM NaCl, pH 6.5) were incubated with doxorubicinhydrochloride (drug/phospholipid ratios of 140-170 mg/mmol) at 60° C.for 45 min followed by quenching on ice and removal of unencapsulateddoxorubicin by gel chromatography. Doxorubicin was assayed byspectrophotometry (Example 71), and phospholipid was assayed by Bartlettmethod (Example 70). The properties of resulting liposomes aresummarized in Table 30 below.

TABLE 30 Properties of liposomal doxorubicin at various lipidcompositions. Liposome size, drug/ nm (mean ± SD phospholipid Lipidcomposition (molar ratio) by QELS) (mg/mmol) DSPC/Chol/PEG-DSPE(3:2:0.015) 81.8 ± 27.3 163.6 ± 4.4  DSPC/Chol (3:2) 79.1 ± 27.9 137.0 ±17.5 DSPC/Chol/Glu-DSPE (2.85:2:0.15) 83.6 ± 27.2 141.7 ± 10.4DSPC/Chol/PEG-DSPE (2.7:2:0.3) 83.7 ± 23.1 175.0 ± 6.8 

Blood pharmacokinetics of these doxorubicin-containing liposomes havinglipid composition of DSPC/Chol/PEG-DSPE 2.7:2:0.3 was studied in rats ata single i.v. dose of 5 mg doxorubicin/kg as described in Example 9. Theliposomes were long circulating (half-life of about 28 hours) (FIG. 36).The stable doxorubicin-to-phospholipid ratio indicated that theformulation was remarkably stable against the drug leakage in thecirculation, losing less than 25% of the drug over a 48-hour timeperiod.

Example 57. Doxorubicin-Loaded Liposomes and Anti-HER2 ImmunoliposomesPrepared by TEA-Sulfate Method: Preparation and In Vivo AntitumorEfficacy Against HER2-Overexpressing Human Breast Cancer Xenografts

Doxorubicin-loaded liposomes having various lipid compositions andproperties (listed in the table below) were prepared as described inExample 56. Doxorubicin-loaded anti-HER2 immunoliposomes were preparedfrom doxorubicin-loaded liposomes by co-incubation with anti-HER2 scFvF5-PEG-DSPE conjugate (approx. 30 scFv/liposome) as described in Example19. NCR nu/nu mice bearing the subcutaneous human breast tumor xenograft(BT-474) were raised, treated (in groups of 10-12 animals) withliposomal or anti-HER2 immunoliposomal doxorubicin at a dose of 5 mg/kgonce weekly for a total of three weeks once the tumors reached anaverage size of 200 mm³, and the tumor progression and animal bodyweights were monitored as described in Example 29. For non-targeteddoxorubicin liposome formulations, the lipid compositions containing noPEG-DSPE, 0.5 mol. % PEG-DSPE, or 10 mol. % PEG-DSPE, were studied; forF5-immunoliposomal doxorubicin, the formulations with 0.5 mol. %PEG-DSPE and 10 mol. % PEG-DSPE were studied (here the quantity ofPEG-DSPE is expressed as mol. % of liposome phospholipid). The results(FIG. 37, Table 31) demonstrated that all doxorubicin treatments wereeffective in retarding the tumor growth. On the basis of tumor sizes atday 53 post inoculation, the differences in tumor growth inhibitionamong all three non-targeted liposome groups did not raise tostatistical significance (ANOVA p=0.081), but the immunoliposomedoxorubicin was significantly more efficacious than non-targetedliposomal doxorubicin)(ANOVA p=5.5×10⁻¹⁰, the “10% PEG-DSPE” formulationbeing more efficacious than “0.5% PEG-DSPE” (Student's t-test, p=0.027).In the“10% PEG-DSPE” F5-ILs group, the tumors regressed to 1 mm³ or lessin 67% of animals, while in “0.5% PEG-DSPE” F5-ILs group only in 9%. Inthe control group (saline treatment) the tumors exceeded the acceptablesize limit of 15% body weight at day 38-43.

TABLE 31 Liposomal doxorubicin in vivo antitumor efficacy study:liposome characteristics and treatment outcomes. drug/ Average tumorLiposome phospholipid size at day size, nm ratio, mg/mmol 58, mm³ Lipidcomposition (mean ± SD) (mean ± SD) (mean ± SEM) DSPC/Chol/PEG-DSPE 83.4± 23.3 136.7 ± 6.7 490 ± 74 (3:2:0.015) DSPC/Chol (3:2) 80.5 ± 26.6151.2 ± 1.9 587 ± 61 DSPC/Chol/PEG-DSPE 81.0 ± 24.7 140.1 ± 4.2 365 ± 60(2.7:2:0.3) DSPC/Chol/PEG-DSPE not measured 140.7 ± 2.8 119 ± 39(3:2:0.015) + F5 scFv-PEG-DSPE DSPC/Chol/PEG-DSPE not measured 132.9 ±2.2 15.5 ± 7.6 (2.7:2:0.3) + F5 scFv-PEG-DSPE

Example 58 Preparation of Liposomal Vinblastine and BloodPharmacokinetics of Liposomal Vinblastine in Rats

Liposomes with entrapped aqueous TEA-SOS solution (0.65 M TEA, pH 6.4,osmolality 502 mmol/kg) and size 99.5±10.2 nm (mean±SD by QELS) wereprepared by the method of Example 11 using extrusion 2 times through twostacked 0.2 μm polycarbonate membranes and ten times through two stacked0.08 μm polycarbonate membranes. Vinblastine (VBL) in the form ofvinblastine sulfate USP was added at a drug-to-phospholipid ratio of 150mg/mmol. The pH of the drug-liposome mixture was adjusted to 6.5 using 1N NaOH, and the mixture was subsequently incubated at 60° C. for 30 min.The reaction was then cooled on ice for 15 min and unencapsulated drugremoved using Sephadex G-75 gel filtration chromatography, eluting with5 mM HEPES-Na, 135 mM NaCl, pH 6.5. The purified liposomes were thenanalyzed for VBL spectrophotometrically and for phospholipid by Bartlettmethod as in Examples 70 and 71. [³H]-CHE was included in theformulation at a ratio of 1.5 mCi/mmol phospholipid. The liposomalvinblastine had 152.4±12.0 mg VBL/mmol phospholipid (quantitativeencapsulation).

Blood pharmacokinetics of the liposomal vinblastine in female Albinorats (8-9 weeks old; 200 g) at a dose of 5 mg VBL/kg was studied asdescribed in Example 9. Vinblastine was quantified in blood plasmasamples as described in Example 41 (using vinorelbine as internalstandard). Vinblastine liposomes showed good circulation longevity(plasma half-life of the lipid component 12.8±0.04 hours) (FIG. 38) andvery good stability against drug leakage from the liposomes with greaterthan 70% of the initial vinblastine load remaining encapsulated after 24h (FIG. 39). The post-injection time to achieve release of 50% of theencapsulated drug was found to be 40.6±1.2 hours.

Example 59 Preparing Liposomes Loaded with Vincristine Using TEA-SOSMethod and the Effect of pH on the Loading Efficiency

Liposomes with the size of 86.6±12.9 nm (by QELS), lipid composition ofDSPC/Chol/PEG-DSPE in the molar ratio of 3:2:0.015 and entrapped aqueousTEA-SOS solution (0.65 M TEA, pH 5.4, osmolality 521 mmol/kg) wereprepared by the method of Example 11 using extrusion step of 15 passagesthrough two stacked 0.08 μm pore size polycarbonate membranes.Vincristine (VCR) was added to the liposomes in 5 mM HEPES-Na, 5%dextrose aqueous buffer, pH 6.5, as vincristine sulfate at adrug-to-phospholipid ratio of 350 μg vincristine/μmol phospholipid, thepH was adjusted to the indicated ratio using 1 N NaOH, the mixture wasincubated at 60° C. for 30 min, chilled on ice for 15 min, and theliposomes were separated from unencapsulated drug using Sephadex G-75gel filtration chromatography, eluting with HBS-6.5 (20 mM HEPES, 135 mMNaCl, pH 6.5). The purified liposomes were then analyzed for vincristineby spectrophotometry using absorbance at 265 nm after solubilization inacid isopropanol, and for the phospholipid content using the phosphateassay of Bartlett (1959).

The results are shown below in Table 32. The drug loading was in excessof 90% in the range of pH 4.5-7.5, and practically quantitative at pH5.0-7.5. At pH 3.5, which is the pH observed in the liposome mixtureafter addition of the drug, but without pH adjustment, the loading wasconsiderably lower.

TABLE 32 pH-Dependence of vincristine loading into liposomes withentrapped TEA-SOS. Drug/phospholipid Loading pH ratio, μg/μmolefficiency (%) 3.5 39.7 ± 4.9  11.3 ± 0.2 4.5 327.2 ± 20.6  93.5 ± 5.45.0 360.6 ± 5.8  103.0 ± 1.7 5.5 371.2 ± 30.2 106.1 ± 9.1 6.0 347.7 ±20.4  99.3 ± 5.8 6.5 347.7 ± 20.9  99.4 ± 5.9 7.0 377.3 ± 22.2 107.8 ±6.8 7.5 371.5 ± 24.9 106.1 ± 7.6

Example 60. Preparing Liposomes Loaded with Vincristine Using TEA-SOSMethod: Effect of the Drug/Lipid Ratio on the Loading Efficiency

SOS-TEA-containing liposomes were prepared as in Example 59 and loadedwith vincristine sulfate at a drug-to-phospholipid ratio of 150-550 μgvincristine/μmol phospholipid at pH 6.5 according to the procedure ofExample 59. The liposomes purified from unencapsulated drug were thenanalyzed for VCR by spectrophotometry and for the liposome phospholipidusing the assay of Bartlett (1959). The drug loading efficiency was inexcess of 90% over the whole studied range of drug/lipid ratios, and waspractically quantitative between 150-450 μg vincristine/μmolphospholipid (Table 33).

TABLE 33 Vincristine loading into liposomes containing TEA-SOS atdifferent drug-to-lipid ratios. Input drug-to- Encapsulated drug-to-Loading phospholipid (μg/μmol) phospholipid (μg/μmol) efficiency (%) 150163.6 ± 6.6  109.0 ± 4.8 250 251.1 ± 17.0 100.5 ± 6.8 350 347.7 ± 20.9 99.4 ± 5.9 450 452.0 ± 18.8 100.4 ± 4.2 550 521.6 ± 24.9  94.8 ± 4.3

Example 61. Preparing Immunoliposomal Vincristine and Cytotoxicity ofLiposomal and Immunoliposomal Vincristine Against Cancer Cells In Vitro

Liposomal vincristine (Ls-VCR) was prepared as described in Example 59using the drug/phospholipid ratio of 350 mg/mmol. HER2-specificF5-immunoliposomal vincristine (F5-ILs-VCR) was prepared from theliposomal vincristine by co-incubation with anti-HER2 scFv F5-PEG-DSPEconjugate as described in Example 19. “Free” vincristine (VCR) solutionwas prepared by dilution of vincristine sulfate USP in water, followedby sterile filtration. Cytotoxicity of VCR, Ls-VCR, and F5-ILs-VCRagainst HER2-overexpressing human breast carcinoma cells SKBr-3 (ATCC)was determined by MTT-based cell viability assay using the procedure ofExample 27, wherein the cells were inoculated into 96-well microtiterplates at 5,000 cells/well, acclimated overnight, and incubated with thedrug-containing media for 4 hours, followed by post-incubation in adrug-free medium for 3 days. The results are shown on FIG. 40. The IC₅₀was 75 ng/ml for free VCR, 11 ng/ml for F5-ILs-VCR, and 3 μg/ml forLs-VCR. The targeted liposomal vincristine prepared according to theinvention was 6.8 times more active than the free drug, and 273 timesmore active than non-targeted liposomal drug, showing substantialenhancement in anticancer activity as a function of cell-specific drugdelivery.

Example 62 Blood Pharmacokinetics of Ls-VCR in Rats

Liposomes with entrapped SOS-TEA solution (0.65 M TEA, pH 5.8,osmolality 530 mmol/kg), and lipid composition of DSPC/Chol/PEG-DSPE(molar ratio 3:2:0.015), also containing [³H]-CHE at 1.5 mCi/mmolphospholipid, were prepared by the method of Example 11 using extrusionstep of 10 passages through two stacked polycarbonate membranes with thepore size of 80 nm or 100 nm. The liposomes were loaded with VCR at pH6.5, drug/phospholipid ratio of 350 mg/mmol, as described in Example 59.The VCR-loaded liposomes were administered i.v. to female albino rats(180-220 g) at a dose of 5 mg VCR/kg, and the blood pharmacokinetics ofthe drug and the liposome lipid was studied as described in Example 9.The amount of VCR in the blood samples was quantified by HPLC asdescribed in Example 41, except that the volume ratio of aqueoustriethylammonium acetate (pH 5.5) and acetonitrile in the mobile phasewas 65:35. The typical retention time for VCR was 8.8 min. The resultsare shown in FIG. 41 and Table 34. Both preparations had extensivecirculation longevity (blood half-lives of 12-17 hours). Liposomalvincristine was remarkably stable against drug leakage in bothpreparations (half-release time over 120 hours) (FIG. 42).

TABLE 34 Characteristics of liposomes loaded with vincristine at 350mg/mmol phospholipid using TEA-SOS method. Ex- t_(1/2) trusion LiposomeDrug load, t_(1/2β) t_(1/2β) VCR pore size, nm mg/mmol lipid, VCR,release, size, nm (mean ± SD) phospholipid hours hours hours 80 101.2 ±20.2 347.7 ± 20.93 17.5 ± 1.5 16.0 ± 2.0 >120 100 125.6 ± 32.0 366.8 ±18.11 12.1 ± 0.7 12.0 ± 0.8 Not detect- able

Example 63. Blood Pharmacokinetics of Ls-VCR in Rats at VariousDrug/Lipid Ratios

Liposomes with entrapped SOS-TEA solution (0.65 M TEA, pH 6.4,osmolality 485 mmol/kg), and lipid composition of DSPC/Chol/PEG-DSPE(molar ratio 3:2:0.015) also containing [³H]-CHE at 1.5 mCi/mmolphospholipid were prepared by the method of Example 11 using extrusionstep of 10 passages through two stacked polycarbonate membranes with thepore size of 50 nm or 80 nm. The liposomes were loaded with VCR at pH6.5 as described in Example 59 by adding a stock 20 mg/mL aqueous VCRsulfate solution at the calculated drug/lipid ratios of 100, 200, or 350mg/mmol phospholipid. The efficiency of drug loading was over 96% forall preparations. The VCR-loaded liposomes were administered i.v. tofemale albino rats (8-9 week old, 190-220 g) at a dose of 5 mg VCR/kg,and the blood pharmacokinetics of the drug and the liposome lipid wasstudied as described in Example 62. The results are shown in Table 35.Liposomal vincristine had good circulation longevity (blood half-life ofthe drug about 20-30 hours) and was exceptionally stable at all studiedsizes and drug-to-lipid ratios (half-life of drug release over 93hours).

TABLE 35 Characteristics of liposomes loaded with vincristine usingTEA-SOS method at various drug/lipid ratios. Extrusion Liposome VCR,mg/mmol t_(1/2) t_(1/2) t_(1/2) drug pore size, size, nm phospholipidlipid, VCR, release, nm (mean ± SD) added encapsulated hours hours hours50  76.8 ± 27.2 100  96.1 ± 3.0 35.6 ± 2.7 30.3 ± 4.0 227 ± 96 200 193.3± 3.9 20.8 ± 2.2 18.4 ± 0.7  244 ± 130 350  375.2 ± 10.0 24.8 ± 0.9 19.6± 0.9 93.2 ± 6.7 80 101.6 ± 25.3 100 104.5 ± 2.1 33.0 ± 7.6 26.8 ± 4.8153 ± 10

Example 64. Preparation of HER2-Targeted Liposomal Vincristine andAntitumor Efficacy of Non-Targeted and HER2-Targeted LiposomalVincristine Against HER2-Overexpressing Human Breast Cancer Xenograftsin Mice

Vincristine-loaded liposomes (Ls-VCR-SOS) using TEA-SOS method wereprepared according to Example 63 (with omission of [³H]-CHE component)using 50 nm pore size membrane extrusion and drug loading atdrug/phospholipid ratio of 100 mg/mmol. F5 immunoliposomal vincristine(F5-ILs-VCR) was formed by incubating Ls-VCR-SOS with anti-HER2 scFvF5-PEG-DSPE conjugate (Example 19) as described in Example 43.Vincristine-loaded liposomes using TEA-citrate (Ls-VCR-Cit) wereprepared similarly to Ls-VCR-SOS liposomes, except that triethylammoniumcitrate solution (prepared by titrating aqueous citric acid with neattriethylamine to pH 5.1 and adjusting the concentration to 0.65 Mtriethylamine) was substituted for TEA-SOS solution. The treatment studydesign followed the method of Example 10. Subcutaneous xenograft tumorsof BT-474 human breast carcinoma were raised in nude mice, and when thetumors reached the size of 250 mm³ (range 144-309 mm³) the mice in thegroups of eight to nine, were treated with free VCR, Ls-VCR, orF5-ILs-VCR at a weekly i.v. dose of 2 mg VCR/kg for a total of threeweeks, starting at day 19 post tumor inoculation. The tumor sizes andanimal body weights were monitored as described in Example 10. For thecontrol group, mice were treated with an equal volume of saline. Thedifferences in tumor sizes between the treatment groups werestatistically assessed at day 63 post tumor inoculation usingMann-Whitney test. The dynamics of average tumor size in the groups isshown in FIG. 43. F5-ILs-VCR demonstrated maximum efficacy when comparedto either Ls-VCR or free VCR, causing at day 63 complete tumorregressions in six out of eight animals (75%). Ls-VCR-Cit was alsoeffective, causing complete tumor regressions still observed at day 63in two out of nine animals (22%), however, it was less effective thanF5-ILs-VCR (p<0.005). Ls-VCR-SOS and free VCR were equally effective(p>0.2) and less effective than either F5-ILs-VCR or Ls-VCR-Cit. Thus,surprisingly, with cell-targeted delivery, a liposomal drug encapsulatedusing a polyvalent anion of the present invention proved moreefficacious than the drug liposomally encapsulated via non-bindinganion. Animal body weight dynamics showed that all liposomal VCRpreparations were less toxic than free VCR, causing less body weightloss during treatment (FIG. 44).

Example 65. Preparation of EGFR-Targeted Liposomal Vincristine andAntitumor Efficacy of Non-Targeted and EGFR-Targeted LiposomalVincristine Against EGFR-Overexpressing Human Brain Cancer Xenografts inMice

Vincristine-loaded liposomes (Ls-VCR) were prepared using TEA-SOS methodas in Example 64. EGFR-targeted immunoliposomal vincristine was preparedby co-incubation of the liposomes with anti-HER2 Fab′ C225Fab-PEG-DSPEconjugate as described in Example 36.

Male NCR nu/nu mice (5-6 week old, weighing 17-20 g) were injectedsubcutaneously in the flank area with 0.15 ml of the cell growth mediumcontaining 1×10⁷ U87 human glioblastoma cells stably expressingepidermal growth factor receptor (HER1) mutant EGFRvIII. At day 11 whenthe mean tumor size reached 300-400 mm³, the mice were randomly dividedinto four groups of 10-12 animals/group. Treatments with free VCR(vincristine sulfate 1 mg/mL in saline), Ls-VCR, or C225Fab-ILs-VCR ati.v. dose of 1.5 mg/kg were administered on days 11, 18, and 25 posttumor inoculation. Mice in the control group were similarly injectedwith an equal volume of normal saline. The tumor sizes and mouse bodyweights were monitored as in Example 10. The results are shown in FIG.45. All animals treated with VCR formulations showed retardation oftumor growth compared to control animals. There was no significantdifference between the groups treated with free VCR and Ls-VCR.EGFR-targeted C225Fab-ILs-VCR was more efficacious than free ornon-targeted liposomal VCR.

Example 66. Preparation of Liposomes with Entrapped TriethylammoniumInositol Hexaphosphate (TEA-IHP) Solution

A polyanionized polyol, inositol hexaphosphate (IHP) dodecasodium salt,was obtained from Sigma (St. Louis, Mo.). Aqueous solution containing0.65 M triethylammonium and 0.681 M of phosphate groups, pH 6.5, andosmolality of 718 mmol/kg, was prepared by ion-exchange on the Dowex50Wx8-200 cross-linked sulfonated polystyrene resin followed bytitration with neat TEA and dilution with water according to theprocedure of Example 4. The residual sodium content was less than 1% ofthe sum of cations. Dry lipids (150 μmol DSPC, 100 μmol Chol, 0.75 μmolPEG-DSPE) were dissolved in 0.5 ml of 100% ethanol USP at 60° C. andmixed with 4.5 ml of triethylammonium inositol hexaphosphate solutionpre-heated to the same temperature. The ethanol was partially removed byrotary evaporation at 30-40 mm Hg and 40-45° C. until the mixture showedno bubbling. The lipid suspension was then extruded at 60-65° C. 15times through two stacked 0.1 μm pore size polycarbonate membranes. Theresulting liposomes were 104.3±39.0 nm in size by QELS. Theunencapsulated triethylammonium IHP was removed by gel chromatography ona Sepharose 4B column, eluted with 5 mM HEPES-Na, 5% dextrose, pH 6.5buffer, and the liposomes were quantified by phospholipid concentrationusing Bartlett's method with extraction according to Example 70.

Example 67. Loading of Drugs into Liposomes with Entrapped TEA-IHPSolution

The liposomes of Example 67 were loaded with CPT11 or vinorelbineVinorelbine was loaded at a drug-to-phospholipid ratio of 175 or 350g/mol, and CPT11 at a ratio of 250 or 500 g/mol. The drugs were added tothe liposomes in the HEPES-dextrose buffer (Example 67) at the inputdrug/phospholipid ratios, indicated below (see Table 36). If necessary,the pH was adjusted to 6.5-6.8 using 1 N NaOH. The mixtures wereincubated at 60° C. for 30 min, cooled down on ice for 15 min, andchromatographed on a Sephadex G-25 gel filtration column, eluted with 5mM HEPES-Na, 145 mM NaCl, pH 6.5. Aliquots of the purified liposomeswere solubilized in acidified methanol and analyzed by spectrophotometry(Example 71). Phospholipid was quantified by the method of Bartlett(1959) with extraction (Example 70). Both drugs loaded quantitatively(i.e., practically 100%) into the liposomes, as shown below in Table 36.

TABLE 36 Properties of drugs loaded into liposomes with entrappedinositol hexaphosphate. Input Encapsulated drug/lipid drug/lipid ratio,g/mol ratio, g/mol Loading Drug phospholipid phospholipid efficiency, %Vinorelbine 175 175.3 ± 8.0  100.2 ± 4.5 Vinorelbine 350 352.3 ± 11.8100.6 ± 3.3 CPT-11 250 265.1 ± 11.2 106.1 ± 4.7 CPT-11 500 518.7 ± 27.8103.7 ± 5.8

Example 68. Chemical Stability of Free or Liposomal CPT-11 in thePresence of Mouse Plasma In Vitro

In the body, CPT-11, which is a pro-drug, undergoes chemicaltransformation to form an active drug metabolite known as SN-38. BothSN-38 and CPT-11 are also converted from their active lactone forms intoan inactive products known as a SN-38 or CPT-11 carboxylates. In thisExample the effect of liposomalization of CPT-11 in accordance with thepresent invention on the CPT-11 chemical conversion into these productsin the presence of blood plasma was studied. Liposomes with entrappedtriethylammonium sucroseoctasulfate (0.65 M TEA, pH 6.4, osmolality 485mmol/kg) and lipid composition of DSPC, Cholesterol, and PEG-DSPE in amolar ratio of 3:2:0.015 were prepared according to Example 11, usingextrusion ten times through two stacked 0.08 μm polycarbonate filters.The liposomes were 87.4±19.2 nm in size by QELS. CPT-11 was loaded atapproximately 500 mg of CPT-11 base/mmol liposome phospholipid byincubation in an aqueous 5 mM HEPES-Na, 5% dextrose, pH 6.5, at 60° C.for 30 min., followed by quenching on ice for 15 min. The CPT-11-loadedliposomes were then purified on a Sephadex G-75 column eluted with HEPESbuffered saline (5 mM HEPES, 145 mM NaCl, pH 6.5). The resulting CPT-11liposomes had 536.5±20.1 mg CPT-11/mmol of phospholipid. Free CPT-11solution was prepared by freshly dissolving Irinotecan Hydrochloride USPat 1 mg/ml in 144 mM aqueous NaCl, acidified to pH 3 with diluted HCl.Ten-μ1 aliquots of free or liposomal CPT-11 or free CPT-11 were mixedwith 90 μl of heparin-stabilized mouse plasma (Harlan Bioproducts, USA)and incubated at 37° C. in a shaking water bath. At a given time pointliposome samples, in triplicate, were chromatographed on Sepharose CL-4Bsize exclusion columns (2 ml bed volume) eluted with HBS-6.5, and thedrug-containing fractions were detected by fluorescence. The first (voidvolume) and second (trailing) drug-containing peaks were collected andconsidered as the liposomally encapsulated and released drug fractions.The samples were extracted with 400 μl of ice-cold methanol by vortexingfor 10 s followed by centrifugation at 14,100×g for 5 min. Thesupernatants were analyzed for CPT-11 and its conversion products byHPLC using modification of a method by Warner and Burke, J Chromatogr.,Ser. B Biomed. Sci. Appl. 1997, vol. 691, p. 161-71. The mobile phaseconsisted of 3% triethylammonium acetate pH 5.5 (solution A) andacetonitrile (solution B) delivered at 1.0 ml/min in a linear gradientof 20 vol % B to 50 vol. % B in 14 min. The eluted products weredetected by fluorescence with an excitation at 375 nm and emission at500 nm. The retention times were 5.3 min (CPT-11 carboxylate), 6.8 min(SN-38 carboxylate), 9.3 min (CPT-11) and 11.0 min (SN-38). The results(Table 37) indicated that while free CPT-11 and CPT-11 released from theliposomes underwent conversion, intraliposomal CPT-11 was quite stable.

TABLE 37 Conversion of free and liposomal CPT-11 into SN-38 andcarboxylate forms in mouse plasma in vitro. CPT-11, % SN-38, % Time,carbox- carbox- Sample hours lactone ylate lactone ylate Free CPT-11 2 1.9 ± 0.4 35.2 ± 1.9 4.4 ± 0.1 58.4 ± 2.1 12 <0.1 11.5 ± 0.9 9.9 ± 0.878.6 ± 1.3 24 <0.1 <0.1 22.5 ± 9.8  77.5 ± 9.8 Ls-CPT-11 12 97.7 ± 0.1<0.1 2.3 ± 0.1 <0.1 (encap- 24 97.7 ± 0.1 <0.1 2.3 ± 0.1 <0.1 sulated)Ls-CPT-11 12  60.5 ± 10.4 25.0 ± 7.1 5.0 ± 0.3  9.5 ± 3.0 (released) 2478.3 ± 6.7 14.0 ± 5.2 6.5 ± 0.5  1.2 ± 1.7

Example 69. In Vivo Chemical Stability of Free or Liposomal CPT-11 inRats

Liposomal CPT-11 was prepared as in Example 68 using triethylammoniumsucroseoctasulfate having 0.65 M TEA, pH 6.4, and osmolality 502mmol/kg. The liposome size was 98.5±18.4 nm, and CPT-11 encapsulationwas 510.1±16.5 mg CPT-11/mmol phospholipid. The liposomal and freeCPT-11 was administered intravenously at the dose of 25 mg/kg intofemale Albino rats (180-220 g) with indwelling central venous catheters,and the blood samples were withdrawn at intervals over the period of 48hours. The blood samples were mixed with ice-cold PBS containing 0.04%EDTA and quickly centrifuged to remove blood cells. Aliquots of thesupernatant fluids were assayed for CPT-11, SN-38, and their carboxylateforms by HPLC as in Example 68 above. The results are shown in FIGS. 46and 47. Whereas the free CPT-11 was cleared very rapidly, beingundetectable after 30 min, the liposomal CPT-11 was persistent in thecirculation (t_(1/2) 15.2 hours) with 37.8% of the drug in the blood at24 h, and approximately 10% of the drug still in the circulation after48 h. There was no detectable conversion of the liposomal form of CPT-11to either SN-38 or the carboxylate form of CPT-11. Free CPT-11, i.e.administered as a solution, cleared from the circulation quite fast(half-life of about 16 min), and there was appreciable conversion to thecarboxylate form of the drug.

Example 70. Quantification of the Liposome Phospholipid

Modified acid digestion—blue phosphomolybdate method I. This method ismodified after Bartlett (1959). 10-20 ml aliquots of liposomes areplaced into heat-resistant glass tubes, digested by heating with 0.5 mlof 10 N sulfuric acid for 2 hours at 110-130° C., mineralized byaddition of 50 ml of 9% hydrogen peroxide, and heated for additional 30min. until no hydrogen peroxide is detected by an indicator paper strip.The digested samples at ambient temperature are diluted with 1 ml of0.2% aqueous ammonium molydbate, mixed with 0.1 ml of 5% aqueousascorbic acid, and incubated on a boiling water bath for 10 min. Theabsorbance of reduced phosphomolybdate complex is measured at 800 nm andcompared to a standard curve concurrently produced using inorganicphosphate standard solutions.

Modified acid digestion—blue phosphomolybdate method II. This method isa modification of the method by Morrison (1964). 5 μl aliquots ofliposomes having 1-10 mM phospholipid are mixed with 60 μl ofconcentrated sulfuric acid and 10 μl of 30% hydrogen peroxide inheat-resistant glass tubes. The mixtures are heated at 200-220° C. for10 min., diluted with 0.7 μl of deionized water, mixed with 10 μl of 10%aqueous sodium sulfite, incubated on a boiling water bath for 5 min, andchilled down to ambient temperature. 200 μl of 2% aqueous ammoniummolybdate and 10 μl of 10% aqueous ascorbic acid are added, and thesamples are incubated on a boiling water bath for 10 min. Samples arequickly chilled to ambient temperature, and the absorbance of reducedphosphomolybdate complex is determined at 825 nm against the blanksample. The amount of phospholipid is determined from the standard curveobtained in the same run using standard solutions having 2, 4, 6, 8, and10 mM potassium dihydrogen phosphate.

Extraction method. 25-100 μl aliquots of liposomes are extracted 3 timeswith 200 μl portions of methanol-chloroform mixture (1:2 by volume). Theorganic phases are combined in a heat-resistant glass tube, and thesolvents are removed in vacuum. The residues are treated with 10Nsulfuric acid and further assayed for phosphorus according to the methodI above.

Unless indicated otherwise, the analytical data are presented as themean±standard error of triplicate runs.

Example 71. Quantification of Drugs in the Liposomes

Spectrophotometric quantification. Aliquots of liposomes (10-50 μl) aremixed with 1 mL of 70 vol. % aqueous isopropanol containing 0.075-0.1 NHCl, and the absorbance against the blank sample is measured at thefollowing wavelengths: doxorubicin, 485 nm; CPT-11 and topotecan, 372nm; ellipticines, 306 nm, vinorelbine, 270 nm; vincristine andvinblastine, 265 nm. The amount of drug is determined by comparison to aconcurrently run standard curve.

Fluorometric quantification. Aliquots of liposome-containing samples(e.g., blood plasma) are diluted with acidified isopropanol (0.02-0.1 mlaliquots: 1 mL of 70% isopropanol-0.075 N HCl; >0.1 ml aliquots: 90%isopropanol-0.1 N HCl to 1 mL). If protein precipitation occurs, thesamples are incubated on ice 1-2 hours and clarified by centrifugation10 min at 12,100×g. The fluorescence of the supernatants is measured atthe following wavelengths: CPT-11, excitation 370 nm, emission 423-425nm; Topotecan, excitation 380-385 nm, excitation 520-525 nm;ellipticines, excitation 306 nm, emission 520 nm. The amount of drug iscalculated from concurrently run standard curves after subtraction ofthe blank fluorescence.

Example 72. Effect of Lipopolymers on the Loading Efficiency ofVinorelbine into Liposomes

Liposomes composed of DSPC 200 molar parts, cholesterol 133 molar parts,and poly(ethylene glycol)(mol. weight 2,000)-derivatized lipids PEG-DSPE(1-20 molar parts) or PEG-DSG (20 molar parts), and containingencapsulated 0.65 M TEA-SOS solution were prepared according to themethod of Example 11, using 80 nm pore size membrane for extrusion step.The liposomes were loaded with vinorelbine at the drug/phospholipidratio of 350 mg/mmol and purified from unencapsulated drug according tothe method of Example 40. The liposomes were assayed for drug and lipidcontent as described in Examples 70, 71, and for the liposome size byQELS using volume-weighted Gaussian approximation. The results (Table38) indicated that while anionic PEG derivative, PEG-DSPE, at the amountof more than 1 mole % of the liposome phospholipid (0.3 mole % of thetotal lipid), had negative effect on the drug loading efficiency, theneutral derivative, PEG-DSG, surprisingly, did not affect the loadingefficiency even at 9.1 mole % of the liposome phospholipid (5.7 mole %of total lipid).

TABLE 38 Properties of vinorelbine liposomes prepared by TEA- SOS methodat various amounts of PEG-lipid derivatives. PEG-lipid Loading amount,Liposome Drug load, efficiency, mol. % of size, nm mg/mmol % encap-PEG-lipid total lipid (mean SD) phospholipid sulation PEG-DSPE 0.3 108 ±32 359.5 ± 17.8 102.7 ± 5.2  PEG-DSPE 0.6 110 ± 18 346.6 ± 14.5 99.0 ±4.1 PEG-DSPE 1.8 104 ± 35 332.0 ± 14.0 94.9 ± 3.8 PEG-DSPE 2.9  94 ± 33259.8 ± 9.5  74.2 ± 2.0 PEG-DSPE 4.0 100 ± 36 155.4 ± 7.0  44.4 ± 0.9PEG-DSPE 5.7 103 ± 31 61.2 ± 5.2 17.5 ± 0.3 PEG-DSG 5.7  97 ± 36 362.7 ±14.2 103.6 ± 4.2 

Example 73. Effect of Intraliposomal Drug-Trapping Agent on the BloodLongevity of CPT-11 in Mice

Liposomes with entrapped 0.65 N solutions of triethylammonium (TEA) ortriethanolammonium (TEOA) salts of inositol hexaphosphate (IHP, phyticacid) or sucrose octasulfate were prepared and loaded with CPT-11 at 500g/mol phospholipid following general procedure of Example 66. Theliosomes were administered intravenously to Swiss-Webster mice in thedose of 5 mg CPT-11/kg body weight. Twenty four hours later, the micewere anesthetized, and exsanguinated via open heart puncture. The bloodwas collected, analysed for CPT-11 content in the blood plasma by HPLCas described in Example 68, and the drug amount was expressed as % ofinjected dose remaining in the blood (% ID). TEOA-IHP was less effectivein improving the blood longevity of the drug than TEA-IHP, TEOA-SOAS,and TEA-SOS (Table 39).

TABLE 39 CPT-11 remanence in the blood 24 hours following intravenousadministration of CPT-11 liposomes in mice. Intraliposomal % IDremaining drug-trapping agent in the blood TEOA-IHP 2.74 ± 0.54 TEA-IHP5.86 ± 0.20 TEOA-SOS 7.03 ± 0.17 TEA-SOS 11.32 ± 0.46 

Example 74. Drug Loading into Liposomes Containing 1.05 NDiethylammonium Sucrose Octasulfate

Aqueous solution of 1.05 N diethylammonium sucrose octasulfate (DEA-SOS)pH 6.0, osmolarity 727 mmol/kg, was prepared usingion-exchange/titration method of Example 6 using neat diethylamine(99.5% purity). The lipid matrix of 3 molar parts DSPC, 2 molar partsCholesterol, and 0.015 molar parts PEG2000-DSPE, was formulated intoliposomes (volume-weighted average size 92.4 nm) in the presence ofDEA-SOS solution, and CPT-11 was loaded in the liposomes as variousdrug/lipid input ratios using the method of Example 11. Non-encapsulateddrug was removed by gel-chromatography, and the amount of encapsulateddrug per unit lipid (drug/lipid output ratio) was determined.Encapsulation efficiency was calculated as % of drug/lipid output ratiorelative to input ratio. The results are shown in Table 40. The loadingachieved it's maximum level of about 1.76 mol drug per mol phospholipid(1.67-1.70 mol drug/g total lipid), which is in good agreement with theamount (1.78 mol diethylammonium/mol phospholipid) based on thediethyammonium content of the liposomes, assuming stiochiometricexchange of intraliposomal diethylammonium ions for the drug moleculesand estimated intraliposomal entrapped volume of approximately 1.71/molphospholipid.

TABLE 40 Loading of CPT-11 in DSPC/Chol/PEG-DSPE liposomes containing1.05N DEA-SOS. Drug/lipid input Drug/lipid output Encapsulation ratio,mol/g ratio, mol/g efficiency, % 1.25 1.247 ± 0.038 99.8 ± 3.0 1.501.534 ± 0.052 102.3 ± 3.5  1.80 1.669 ± 0.043 92.7 ± 2.4 2.06 1.690 ±0.054 82.0 ± 2.6 2.20 1.704 ± 0.062 77.5 ± 2.8 2.42 1.685 ± 0.103 69.6 ±4.3

Unless indicated otherwise, the analytical data are presented as themean±standard error of triplicate runs. The rat plasma pharmacokineticdata are the mean±standard error of duplicate runs.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the scope of the invention should be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. The disclosures of allcited articles and references, including patent applications andpublications, are incorporated herein by reference for all purposes.

1-20. (canceled)
 21. A pharmaceutical composition comprising irinotecanliposomes encapsulating irinotecan and a polyphosphate having 13-18phosphate units per polyphosphate molecule, said composition having amolar ratio of irinotecan to total lipids ranging from 0.15:1 to 1.5:1.22. The composition of claim 21, comprising a lecithin and cholesterol.23. The composition of claim 22, wherein the lecithin:cholesterol molarratio is 3:2.
 24. The composition of claim 21, comprising DSPC andcholesterol in a 3:2 molar ratio.
 25. The composition of claim 21,comprising DSPC, cholesterol, and an amphipathic polymer comprising apolyethylene glycol-lipid.
 26. The composition of claim 25, comprising apolyethylene glycol phosphatidylethanolamine.
 27. The composition ofclaim 26, wherein the polyethylene glycol phosphatidylethanolaminecomprises a poly(ethylene glycol) moiety having a molecular weightranging from about 250 to about 20,000.
 28. The composition of claim 21,comprising DSPC, cholesterol, and N-(methoxy-poly(ethyleneglycol)-oxycarbonyl)-distearoylphosphatidylethanolamine, in a molarratio of 3:2:0.015.
 29. The composition of claim 21, comprising DSPC,cholesterol, and methoxy-PEG (2000)-DSPE, in a molar ratio of 3:2:0.015.30. The composition of claim 21, comprising one or more phospholipids,and wherein the liposomes encapsulate irinotecan and a polyphosphatehaving 13-18 phosphate units per polyphosphate molecule, and the ratioof grams of irinotecan to moles of total phospholipids is (134.4±9.3):1.31. The composition of claim 21, comprising one or more phospholipids,and wherein the liposomes encapsulate irinotecan and a polyphosphatehaving 13-18 phosphate units per polyphosphate molecule, and the ratioof grams of irinotecan to moles of total phospholipids is (162.4±4.0):1.